Triazole compounds as ksp inhibitors

ABSTRACT

The present invention provides triazole compounds of Formula I: 
     
       
         
         
             
             
         
       
         
         
           
             as further described herein. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, and a method of treating a disorder mediated, at least in part, by KSP in a mammalian patient comprising administering to a mammalian patient in need of such treatment a therapeutically effective amount of a compound of Formula I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.provisional application Ser. No. 61/324,651, filed on 15 Apr. 2010,which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

This invention generally relates to triazole compounds andpharmaceutically acceptable salts, esters, or prodrugs thereof. Thisinvention is further directed to compositions of such compounds togetherwith pharmaceutically acceptable carriers, to uses of such compounds, totheir preparation, and to related intermediates.

BACKGROUND

Kinesins are motor proteins that use adenosine triphosphate to bind tomicrotubules and generate mechanical force. Kinesins are characterizedby a motor domain having about 350 amino acid residues. The crystalstructures of several kinesin motor domains have been resolved.

Currently, about one hundred kinesin-related proteins (KRP) have beenidentified. Kinesins are involved in a variety of cell biologicalprocesses including transport of organelles and vesicles, andmaintenance of the endoplasmic reticulum. Several KRPs interact with themicrotubules of the mitotic spindle or with the chromosomes directly andappear to play a pivotal role during the mitotic stages of the cellcycle. These mitotic KRPs are of particular interest for the developmentof cancer therapeutics.

Kinesin spindle protein (KSP) (also known as Eg5, HsEgS, KNSL1, orKIF11) is one of several kinesin-like motor proteins that are localizedto the mitotic spindle and known to be required for formation and/orfunction of the bipolar mitotic spindle.

In 1995, the depletion of KSP using an antibody directed against theC-terminus of KSP was shown to arrest HeLa cells in mitosis withmonoastral microtubule arrays (Blangy et al., Cell 83:1159-1169, 1995).Mutations in bimC and cut7 genes, which are considered to be homologuesof KSP, cause failure in centrosome separation in Aspergillus nidulans(Enos, A. P., and N. R. Morris, Cell 60:1019-1027, 1990) andSchizosaccharomyces pombe (Hagan, I., and M. Yanagida, Nature347:563-566, 1990). Treatment of cells with either ATRA (alltrans-retinoic acid), which reduces KSP expression on the protein level,or depletion of KSP using antisense oligonucleotides revealed asignificant growth inhibition in DAN-G pancreatic carcinoma cellsindicating that KSP might be involved in the antiproliferative action ofall trans-retinoic acid (Kaiser, A., et al., J. Biol. Chem. 274,18925-18931, 1999). Interestingly, the Xenopus laevis Aurora-relatedprotein kinase pEg2 was shown to associate and phosphorylate X1Eg5(Giet, R., et al., J. Biol. Chem. 274:15005-15013, 1999). Potentialsubstrates of Aurora-related kinases are of particular interest forcancer drug development. For example, Aurora 1 and 2 kinases areoverexpressed on the protein and RNA level and the genes are amplifiedin colon cancer patients.

The first cell permeable small molecule inhibitor for KSP, “monastrol,”was shown to arrest cells with monopolar spindles without affectingmicrotubule polymerization as do conventional chemotherapeutics such astaxanes and vinca alkaloids (Mayer, T. U., et al., Science 286:971-974,1999). Monastrol was identified as an inhibitor in phenotype-basedscreens and it was suggested that this compound may serve as a lead forthe development of anticancer drugs. The inhibition was determined notto be competitive in respect to adenosine triphosphate and to be rapidlyreversible (DeBonis, S., et al., Biochemistry, 42:338-349, 2003; Kapoor,T. M., et al., J. Cell Biol., 150:975-988, 2000).

In light of the importance of improved chemotherapeutics, there is aneed for KSP inhibitors that are effective in vivo inhibitors of KSP andKSP-related proteins.

SUMMARY OF THE INVENTION

In one embodiment, this invention is directed to substituted triazolecompounds and the pharmaceutically acceptable salts, esters, or prodrugsthereof, their preparation, pharmaceutical compositions, and uses fortreating KSP mediated diseases, wherein the compounds are represented bythe general Formula:

wherein,

R¹ can be selected from C₁₋₆ alkoxy-C₁₋₄-alkyl, C₁₋₆ straight chainalkyl, C₃₋₆ branched alkyl, and —C₃₋₆ cyclo alkyl;

R² is selected from H, and C₁₋₆ straight chain alkyl;

R³ represents —(CH₂)₀₋₃ substituted or unsubstituted pyrrolidinyl or anoptionally substituted C₃₋₅ alkyl;

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH,

—C(O)-unsubstituted morpholinyl, and —C(O)-morpholinyl substituted withup to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms.

In certain embodiments of these compounds of Formula I:

R¹ is selected from C₁₋₆ straight chain alkyl, C₃₋₆ branched alkyl, and—C₃₋₆ cyclo alkyl;

R² is selected from H, and C₁₋₆ straight chain alkyl;

R³ represents —(CH₂)₀₋₃ substituted or unsubstituted pyrrolidinyl;

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH,

—C(O)-unsubstituted morpholinyl, and —C(O)-morpholinyl substituted withup to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms.

In other embodiments, R¹ is C₁₋₆alkoxy-C₁₋₄alkyl, such asmethoxy-substituted C₁₋₄ alkyl and 2-methoxy-2-propyl.

The invention also provides methods for making and for using thesecompounds, and pharmaceutical compositions containing these compounds,as further described herein.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Compounds of the invention include those of Formula (I) or apharmaceutically acceptable salt, ester, or prodrug thereof:

wherein:

R¹ is selected from C₁₋₆alkoxy-C₁₋₄alkyl, C₁₋₆ straight chain alkyl,C₃₋₆ branched alkyl, and C₃₋₆ cyclo alkyl;

R² is selected from H, and C₁₋₆ straight chain alkyl;

R³ represents —(CH₂)₀₋₃— substituted or unsubstituted pyrrolidinyl, suchas —CH₂-pyrrolidinyl;

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-unsubstituted morpholinyl, and —C(O)-morpholinylsubstituted with up to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms.

In compounds of Formula I, R¹ is often a branched-chain alkyl or analkoxy alkyl, and R² is often H. In a preferred embodiment, the compoundof Formula I can be this isomer:

In these compounds, R⁵ is often benzyl, which is optionally substitutedwith up to 3 halogen atoms on the phenyl ring of the benzyl group. R⁵can be unsubstituted; when it is substituted, it is often substitutedwith one or two fluorine atoms.

R⁶ is typically an optionally substituted phenyl ring; in someembodiments, it is phenyl substituted by 1-2 halo groups. In preferredembodiments, R⁶ is fluorophenyl or difluorophenyl, particularly2,5-difluorophenyl.

In certain embodiments of these compounds of Formula I, R¹ is selectedfrom C₁₋₆ straight chain alkyl, C₃₋₆ branched alkyl, and —C₃₋₆ cycloalkyl.

A specific embodiment of the present invention provides a compound ofFormula I, wherein:

R¹ is selected from C₁₋₆alkoxy-C₁₋₄alkyl, preferably methoxy-substitutedC₁₋₄alkyl;

R² represents H;

R³ represents —(CH₂)₁₋₃-substituted pyrrolidinyl;

-   -   R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,        —C(O)-morpholinyl substituted with up to three alkyl groups;

R⁵ represents benzyl, or benzyl substituted with up to two fluoro atoms;and

R⁶ is selected from phenyl substituted with up to two halogen atoms.

A further preferred embodiment of the present invention provides acompound of

Formula I, wherein:

R¹ is selected from C₃₋₆ branched alkyl;

R² represents H;

R³ represents —(CH₂)₁₋₃-substituted pyrrolidinyl;

R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-morpholinyl substituted with up to three alkyl groups;

R⁵ represents benzyl, or benzyl substituted with up to two fluoro atoms;and

R⁶ is selected from phenyl substituted with up to two halogen atoms.

A further preferred embodiment of the present invention provides acompound of

Formula I, wherein:

R¹ represents t-butyl;

R³ represents —(CH₂)-fluoro-pyrrolidinyl;

R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-2,6-dimethyl morpholinyl;

R⁵ represents benzyl, or benzyl substituted with one fluoro atom; and

R⁶ is selected from phenyl substituted with up to two fluoro atoms.

A further preferred embodiment of the present invention provides acompound of

Formula I, wherein:

R¹ represents methoxy-C₁₋₄alkyl;

R³ represents —(CH₂)-fluoro-pyrrolidinyl;

R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-2,6-dimethyl morpholinyl;

R⁵ represents benzyl, or benzyl substituted with one fluoro atom; and

R⁶ is selected from phenyl substituted with up to two fluoro atoms.

Another preferred embodiment provides a compound of Formula I wherein,

R³ represents —(CH₂)₁₋₃-fluoro-pyrrolidinyl; and

R⁴ represents —C(O)-2-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-2,6-dimethyl morpholinyl. In particularly preferred embodiments,R⁴ is selected from:

These R4 groups can be racemic or optically active; in preferredembodiments, R4 is an optically active group having the absolutestereochemistry depicted here. Typically, it is one enantiomer and issubstantially free of its opposite enantiomer, i.e., the R4 group has anenantiomeric excess of at least 90% and often at least 95%.

Yet another preferred embodiment of the present invention provides acompound of Formula I, wherein:

R³ represents a ((3R,4R)-4-fluoropyrrolidin-3-yl)methyl group

R⁴ is selected from —C(O)—CH(CH₃)—OH, and

In particularly preferred embodiments, R⁴ is selected from:

A further preferred embodiment of the present invention provides acompound of Formula I, wherein:

R⁵ represents

and

R⁶ is

In another embodiment, the invention provides compounds of Formula II:

wherein,

R¹ can be selected from C₁₋₆ alkoxy-C₁₋₄-alkyl, C₁₋₆ straight chainalkyl, C₃₋₆ branched alkyl, and —C₃₋₆ cyclo alkyl;

R³ represents —(CH₂)₀₋₃-substituted or unsubstituted pyrrolidinyl orC₃₋₅ alkyl substituted with up to three groups selected from amino andhalo;

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-unsubstituted morpholinyl, and —C(O)-morpholinylsubstituted with up to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I (preferably F); and

R⁶ is selected from phenyl substituted with up to three halogen atoms,preferably F or Cl or both.

In compounds of Formula II, where R⁵ or R⁶ is substituted, preferredsubstituents are F and Cl.

In compounds of Formula II, R¹ is often a branched-chain alkylsubstituted with an alkoxy group such as methoxy, and R² is often H. Ina preferred embodiment, the compound of Formula II can be this isomerhaving the absolute stereochemistry shown:

In particularly preferred embodiments of the compounds of Formula II, R⁴is selected from:

Preferably, these groups are optically active and have the absolutestereochemistry shown.

Yet another preferred embodiment of the present invention provides acompound of Formula II, wherein R³ represents

These R³ groups can be racemic or optically active; in preferredembodiments, R³ is an optically active group having the absolutestereochemistry depicted here. Typically, it is one enantiomer and issubstantially free of its opposite enantiomer, i.e., the R³ group has anenantiomeric excess of at least 90% and often at least 95%.

In the compounds of Formula II, R⁵ is often benzyl, which is optionallysubstituted with up to 3 halogen atoms on the phenyl ring of the benzylgroup. R⁵ can be unsubstituted; when it is substituted, it is oftensubstituted with one or two fluorine atoms.

In the compounds of Formula II, R⁶ is typically an optionallysubstituted phenyl ring; in some embodiments, it is phenyl substitutedby 1-2 halo groups. In preferred embodiments, R⁶ is fluorophenyl ordifluorophenyl, particularly 2,5-difluorophenyl.

In certain embodiments of these compounds of Formula II:

R¹ is selected from C1-6 alkoxy-C1-4-alkyl, C3-6 branched alkyl, andC3-6 cyclo alkyl;

R³ represents —(CH₂)₀₋₃-substituted pyrrolidinyl such as a((3R,4R)-4-fluoropyrrolidin-3-yl)methyl group or —CH₂—CH₂—CH(NH₂)—CH₂Fwhich is preferably

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-unsubstituted morpholinyl, and —C(O)-morpholinylsubstituted with up to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms.

In certain embodiments of the compounds of Formula II, R¹ ismethoxy-substituted C₁₋₄ alkyl. In preferred embodiments of thesecompounds, R¹ is 2-methoxy-2-propyl.

In compounds of Formula II, R³ can contain a substituted pyrrolidinyl;for example, it can represent —(CH₂)₁₋₂-substituted pyrrolidinyl such asa ((3R,4R)-4-fluoropyrrolidin-3-yl)methyl group. The pyrrolidinyl can beattached at any position of the ring, typically at a carbon atom andpreferably at position 3 when counting the ring nitrogen atom asposition 1. The pyrrolidinyl group can be substituted with groups suchas halo, lower alkyl and lower alkoxy. Preferably, the pyrrolidinyl ringis substituted by at least one halo on a ring carbon atom, generally F;in addition, it is optionally substituted by lower alkyl, typically Meor Et, and optionally the lower alkyl is on N.

In preferred embodiments of any of the above-described compounds ofFormula II, R³ represents —CH₂—CH₂—CH(NH₂)—CH₂F such as

or —(CH₂)₁₋₂-substituted pyrrolidinyl, wherein the group—(CH₂)₁₋₂-substituted pyrrolidinyl can be, for example:

wherein R″ is H, Me, Et, isopropyl, or n-propyl. Preferably, thepyrrolidinyl group has the absolute stereochemical configuration shownhere, i.e., it is a ((3R,4R)-4-fluoropyrrolidin-3-yl)methyl groupwherein R″ is H, Me, Et, isopropyl or n-propyl.

In preferred embodiments of the compounds of Formula II, R⁴ is selectedfrom —C(O)—CH(CH₃)—OH,

In particularly preferred embodiments of these compounds, R⁴ is selectedfrom:

Also preferably in these compounds, R⁵ represents

and in some such embodiments, R⁶ is

A particularly preferred embodiment of the present invention provides acompound of Formula I or II selected from:

-   N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;-   N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;-   (S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;-   (S)—N—((R)-1(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;-   (S)—N—((R)-1(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide;    and-   (S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide.

Another particularly preferred embodiment of the present inventionprovides a compound of Formula I or II selected from:

and the pharmaceutically acceptable salts of these compounds.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I or II, including any of the embodiments of these compoundsdisclosed above, and a pharmaceutically acceptable carrier. A preferredembodiment of this aspect of the invention provides a compositionfurther comprising at least one additional agent for the treatment ofcancer. Provided in a further preferred embodiment is a compositionwherein the additional agent for the treatment of cancer is selectedfrom the group consisting of irinotecan, topotecan, gemcitabine,imatinib, trastuzumab, 5-fluorouracil, leucovorin, carboplatin,cisplatin, docetaxel, paclitaxel, tezacitabine, cyclophosphamide, vincaalkaloids, anthracyclines, rituximab, and trastuzumab.

Provided in yet another aspect of the present invention is a method oftreating a disorder mediated, at least in part, by KSP in a mammalianpatient comprising administering to a mammalian patient in need of suchtreatment a therapeutically effective amount of a composition ofcomprising a compound of Formula I or II including any of theembodiments of these compounds disclosed above. A preferred embodimentprovides method of treating a disorder mediated, at least in part, byKSP in a mammalian patient comprising administering to a mammalianpatient in need of such treatment a therapeutically effective amount ofa composition comprising a compound of any of the embodiments of thecompounds of Formula I or II described above, and at least oneadditional agent for the treatment of cancer. A preferred embodiment ofthis aspect of the invention provides a method of treating a disordermediated, at least in part, by KSP in a mammalian patient wherein thedisorder is a cellular proliferative disease; preferably the cellularproliferative disease is cancer.

A further preferred embodiment of this aspect of the invention providesa method of treating a cellular proliferative disease as disclosedabove, wherein the cellular proliferative disease is cancer selectedfrom a group consisting of lung and bronchus; prostate; breast;pancreas; colon and rectum; thyroid; stomach; liver and intrahepaticbile duct; kidney and renal pelvis; urinary bladder; uterine corpus;uterine cervix; ovary; multiple myeloma; esophagus; acute myelogenousleukemia; chronic myelogenous leukemia; lymphocytic leukemia; myeloidleukemia; brain; oral cavity and pharynx; larynx; small intestine;non-Hodgkin lymphoma; melanoma; and villous colon adenoma.

Yet another preferred embodiment of this aspect of the inventionprovides a method wherein the additional agent for the treatment ofcancer is selected from the group consisting of irinotecan, topotecan,gemcitabine, imatinib, trastuzumab, 5-fluorouracil, leucovorin,carboplatin, cisplatin, docetaxel, paclitaxel, tezacitabine,cyclophosphamide, vinca alkaloids, anthracyclines, rituximab, andtrastuzumab.

A particularly preferred embodiment of the present aspect provides amethod for inhibiting KSP in a mammalian patient, wherein said methodcomprises administering to the patient an effective KSP-inhibitingamount of a compound of Formula I or II according to any of theembodiments described herein. In some embodiments, the method employs acompound of Formula I or II as described above; for example, a compoundof Formula I wherein:

R¹ is selected from C₁₋₆ straight chain alkyl, C₃₋₆ branched alkyl, and—C₃₋₆ cyclo alkyl;

R² is selected from H, and C₁₋₆ straight chain alkyl;

R³ represents —(CH₂)₀₋₃ substituted or unsubstituted pyrrolidinyl;

R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-unsubstituted morpholinyl, and —C(O)-morpholinylsubstituted with up to three alkyl groups;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms.

The patient for such methods is generally a human, and typically hasbeen diagnosed as being in need of such treatment prior to initiatingthese methods.

Another preferred embodiment provides a method comprising administeringto the patient an effective KSP-inhibiting amount of a compound ofFormula I or II according to any of the embodiments described above. Insome embodiments, the method uses a compound of Formula I wherein:

R¹ is selected from C₃₋₆ branched alkyl;

R² represents H;

R³ represents —(CH₂)₁₋₃-substituted pyrrolidinyl;

-   -   R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,        —C(O)-morpholinyl substituted with up to three alkyl groups;

R⁵ represents benzyl, or benzyl substituted with at least two fluoroatoms; and

R⁶ is selected from phenyl substituted with up to two halogen atoms.

A further particularly preferred embodiment provides a method comprisingadministering a compound of Formula I, wherein:

R¹ represents t-butyl;

R³ represents —(CH₂)-fluoro-pyrrolidinyl;

R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-2,6-dimethyl morpholinyl;

R⁵ represents benzyl, or benzyl substituted with one fluoro atom; and

R⁶ is selected from phenyl substituted with up to two fluoro atoms.

A further preferred embodiment provides a method comprisingadministering a compound of Formula II, wherein:

R¹ represents 2-methoxy-2-propyl;

R³ represents —(CH₂)-fluoro-pyrrolidinyl or —CH₂—CH₂—CH(NH₂)—CH₂F;

R⁴ is selected from —C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH,—C(O)-2,6-dimethyl morpholinyl;

R⁵ represents benzyl, or benzyl substituted with one fluoro atom; and

R⁶ is selected from phenyl substituted with up to two fluoro atoms.

Yet another particularly preferred embodiment provides a method ofadministering a compound of Formula I or II to treat conditions such ascancer. The method can use a compound according to any of theabove-described embodiments, including a compound of Formula I or II,wherein:

R³ represents

R⁴ is selected from —C(O)—CH(CH₃)—OH,

R⁵ represents

and

R⁶ is

A specifically preferred embodiment of the methods of treatmentdescribed above provides a method for inhibiting KSP in a mammalianpatient, wherein said method comprises administering to the patient aneffective KSP-inhibiting amount of a compound of Formula I selectedfrom:

-   N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;-   N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;-   (S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;-   (S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;-   (S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide;    and-   (S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide.

Another specifically preferred embodiment provides a method forinhibiting KSP in a mammalian patient, wherein said method comprisesadministering to the patient an effective KSP-inhibiting amount of acompound of Formula I or II selected from:

or a pharmaceutically acceptable salt of one of these compounds.

In another aspect, the invention provides a method to make certaincompounds of Formula I or II and key intermediates for their synthesis.Scheme 2 herein depicts one such method for making a preferredpyrrolidine ring moiety for compounds such as those depicted above. Thesynthetic method for making this fluorinated pyrrolidine comprisesfluorination of a trans-3,4-disubstituted pyrrolidine of Formula V toprovide a cis-fluorinated vinyl pyrrolidine compound of Formula VI, andoxidizing the olefin of the compound of Formula VI to provide analdehyde of Formula VII, as shown below:

These transformations can be done on racemic compounds, or withoptically active compounds; in some embodiments, the compounds ofFormula V or VI or VII is optically active and has the absolutestereochemistry shown here, with an enantiomeric excess of at least 90%.In the compounds of Formulas V-VII, R is H or an optionally substitutedC1-C6 alkyl or aryl, and PG represents a protecting group suitable foruse on an aliphatic nitrogen atom. In some embodiments, R is H, and thecompound of Formula V can be prepared from an epoxide of a protected3-pyrroline (see compound 2.3 in Scheme 2) by reaction of the epoxidewith a Grignard reagent, for example. The trans-hydroxy group of FormulaV can be converted into the cis-fluoro group in Formula VI using anysuitable reagent that provides an S_(N)2 exchange to achieve inversionof the chiral center. In some embodiments, this is accomplished using afluoride source in an inert solvent, and a reagent that activates thehydroxyl to make it a suitable leaving group. For example a fluoridesalt such as trialkylamine trihydrofluoride (e.g.,Et₃N-trihydrofluoride) or HF-pyridine can be used in a suitable solventinert to the reaction conditions along with an alkyl or aryl sulfonylfluoride, such as C1-C6 perfluoralkyl sulfonyl fluoride to convert thehydroxyl to F with stereochemical inversion.

The vinylogous group in the compounds of Formula VI can be oxidized toan aldehyde using various conventional methods such as treatment withosmium tetroxide and sodium metaperiodate, or using ozone, to providethe compound of Formula VII. Methods for this transformation are knownin the art.

The compound of Formula VII can then be incorporated into a compound ofFormula I by various methods, including a reductive amination reactionas described herein; or various nucleophilic addition reactions known inthe art for use with such aldehydes and nucleophilic carbon groupssuitable for the desired target compound. In one embodiment, thecompound of Formula VII is attached via reductive amination to acompound of Formula Ia as shown in Scheme 3 to provide a compound ofFormula Ib as illustrated below. The compound of Formula Ia wherein R³is H is optionally protected if it includes a group such as a free amineor hydroxyl requiring protection.

Suitable protecting groups (PG) for use in these reactions andintermediates include amides (e.g., formamide, acetamide,trichloroacetamide) and carbamates (e.g., methyl, ethyl, trichloroethyl,t-butyl, or benzyl carbamate). The amides and carbamates are of generalformula —C(O)-L-A, wherein L is a bond (for amides) or —O— (forcarbamates), and A is an optionally substituted alkyl (C1-6 preferably)or aryl (preferably phenyl); or A can be H when L is a bond.

In some embodiments, this method further comprises reductive aminationof the compound of Formula VII with a compound of Formula Ia:

wherein:

R¹ is selected from C₁₋₆alkoxy-C₁₋₄alkyl, C₁₋₆ straight chain alkyl,C₃₋₆ branched alkyl, and —C₃₋₆ cyclo alkyl;

R² is selected from H, and C₁₋₆ straight chain alkyl;

R⁵ is selected from substituted or unsubstituted benzyl, wherein thesubstituents are selected from Cl, F, Br, and I; and

R⁶ is selected from phenyl substituted with up to three halogen atoms;

to provide a compound of Formula Ib:

In some embodiments, any of the foregoing methods of synthesis furthercomprises synthesizing the compound of Formula V from an epoxide ofFormula IV,

by opening the epoxide with an organometallic reagent of the formula

wherein R is H or an optionally substituted alkyl or aryl group, and Mis a metallic group selected from Li, MgX, and ZnX, where X is ahalogen, to provide the compound of Formula V. For this step, apreferred organometallic reagent is:

wherein X is Cl, Br or I.

The invention thus provides novel intermediates of Formula VI and VII asdescribed above, as well as methods to use these intermediates formaking compounds of Formula I or Ib.

The compounds used in and produced by these methods may be racemic, orany of the compounds having at least one chiral center can be separatedinto single enantiomers or single diastereomers as appropriate. In thepresent invention, it is sometimes preferably to separate the twoenantiomers of the compound of Formula V or VI in order to use a singleenantiomer for making compounds of Formula I or Ib. In some embodiments,the compound of Formula V or VI is made in racemic form, and is thenseparated by chiral chromatography or other conventional means toprovide an optically active compound, preferably essentially free of itsenantiomer. In a preferred embodiment, the compound of Formula V or VIis optically active and is of the specific absolute stereochemistrydepicted herein. In some such embodiments, it is essentially free of theopposite enantiomer.

Representative Compounds of the Invention

Specific compounds within the scope of this invention are exemplified inTable 1 in the Experimental section.

B. Definitions and Overview

As discussed above, the present invention is directed in part to newsubstituted triazole compounds.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention. It must be noted that as usedherein and in the claims, the singular forms “a,” and “the” includeplural referents unless the context clearly dictates otherwise. In thisspecification and in the claims which follow, reference will be made toa number of terms which shall be defined to have the following meanings:

As used herein, “alkyl” or “straight chain alkyl” refers to monovalentsaturated aliphatic hydrocarbyl groups having from 1 to 6 carbon atomsand more preferably 1 to 3 carbon atoms. This term is exemplified bygroups such as methyl, ethyl, n-propyl, n-pentyl and the like.

The term “branched alkyl” as used herein refers to a monovalentsaturated branched alkyl group having from 3 to 6 carbon atoms. Thisterm is exemplified by groups such as i-butyl, i-propyl, t-butyl, andthe like.

“Cyclo alkyl” refers to a alkyl group having from 3 to 6 carbon atomsand wherein three or more carbon atoms are connected to each other so asto form a cycli structure. Illustrative examples include cyclo propyl,cyclo butyl, cyclo pentyl, and cyclo hexyl group.

“Alkoxyalkyl” as used herein refers to an alkyl group that issubstituted with at least one alkoxy group. If not otherwise described,the alkoxyalkyl group comprises up to 10 carbon atoms in the alkoxygroup, and up to 10 carbon atoms in the alkyl group. It attaches to thebase molecule through the alkyl group. In some instances, these groupsare described according to the number of carbon atoms in the alkoxygroup and/or in the alkyl group, such as for example C₁₋₆alkoxy-C₁₋₄-alkyl, which refers to an alkyl group having 1-4 carbonatoms, which is substituted with a C1-C6 alkoxy group. Suitablealkoxyalkyl groups include methoxymethyl; methoxyethyl; ethoxymethyl;ethoxyethyl; methoxypropyl; and methoxy-isopropyl (2-methoxy-2-propyl).

“Halo” or “halogen” refers to fluoro, chloro, bromo and/or iodo andpreferably is fluoro or chloro.

“Biological activity” as used herein refers to an inhibitionconcentration when tested in at least one of the assays outlined in anyof Examples 12-14 and as defined in at least one example thereof.

As used herein, the term “pharmaceutically acceptable salts” refers tothe nontoxic acid or alkaline earth metal salts of the compounds ofFormula (I) or (II). These salts can be prepared in situ during thefinal isolation and purification of the compounds of Formula (I) and(II) or by separately reacting the base or acid functions with asuitable organic or inorganic acid or base, respectively. Representativesalts include, but are not limited to, the following: acetate, adipate,alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate,cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemi-sulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,nicotinate, 2-napth-alenesulfonate, oxalate, pamoate, pectinate,persulfate, 3-phenylproionate, picrate, pivalate, propionate, succinate,sulfate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate.Also, the basic nitrogen-containing groups can be quaternized with suchagents as alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides, and iodides; dialkyl sulfates like dimethyl,diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides like benzyl and phenethyl bromides, and others. Water oroil-soluble or dispersible products are thereby obtained.

Examples of acids that may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulfuric acid and phosphoric acid and such organicacids as oxalic acid, maleic acid, methanesulfonic acid, succinic acidand citric acid. Basic addition salts can be prepared in situ during thefinal isolation and purification of the compounds of Formula (I) or (II)or separately by reacting carboxylic acid moieties with a suitable basesuch as the hydroxide, carbonate or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia, or an organic primary,secondary or tertiary amine Pharmaceutically acceptable salts include,but are not limited to, cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, aluminumsalts and the like, as well as ammonium, quaternary ammonium, and aminecations, including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Other representative organicamines useful for the formation of base addition salts includediethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazineand the like.

As used herein, the term “pharmaceutically acceptable ester” refers toesters which hydrolyze in vivo and include those that break down in thehuman body to leave the parent compound, a salt thereof, or apharmaceutically active metabolite. Suitable ester groups include, forexample, those derived from pharmaceutically acceptable aliphaticcarboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic andalkanedioic acids, in which each alkyl or alkenyl moiety advantageouslyhas not more than 6 carbon atoms. Representative examples of particularesters include, but are not limited to, formates, acetates, propionates,butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrug” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound or a pharmaceuticallyactive metabolite of the above formula, for example by hydrolysis inblood. A discussion is provided in T. Higuchi and V. Stella, PRO-DRUGSAS NOVEL DELIVERY SYSTEMS, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., BIOREVERSIBLE CARRIERS IN DRUG DESIGN, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

As used herein “anticancer agents” or “agent for the treatment ofcancer” refers to agents that include, by way of example only, agentsthat induce apoptosis; polynucleotides (e.g., ribozymes); polypeptides(e.g., enzymes); drugs; biological mimetics; alkaloids; alkylatingagents; antitumor antibiotics; antimetabolites; hormones; platinumcompounds; monoclonal antibodies conjugated with anticancer drugs,toxins, and/or radionuclides; biological response modifiers (e.g.interferons and interleukins, etc.); adoptive immunotherapy agents;hematopoietic growth factors; agents that induce tumor celldifferentiation (e.g. all-trans-retinoic acid, etc.); gene therapyreagents; antisense therapy reagents and nucleotides; tumor vaccines;inhibitors of angiogenesis, and the like. Numerous other agents are wellwithin the purview of one of skill in the art.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves are not intended for inclusion herein. In such cases, themaximum number of such substituents is three. For example, serialsubstitutions of substituted aryl groups with two other substituted arylgroups are limited to -substituted aryl-(substituted aryl)-substitutedaryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxy group alpha to ethenylic or acetylenicunsaturation). Such impermissible substitution patterns are well knownto the skilled artisan.

Compounds of this invention may exhibit stereoisomerism by virtue of thepresence of one or more asymmetric or chiral centers in the compounds.The present invention contemplates the various stereoisomers andmixtures thereof. Certain of the compounds of the invention compriseasymmetrically substituted carbon atoms. Such asymmetrically substitutedcarbon atoms can result in the compounds of the invention comprisingmixtures of stereoisomers at a particular asymmetrically substitutedcarbon atom or a single stereoisomer. As a result, racemic mixtures,mixtures of diastereomers, single enantiomer, as well as singlediastereomers of the compounds of the invention are included in thepresent invention. The terms “S” and “R” configuration, as used herein,are as defined by the IUPAC 1974 “RECOMMENDATIONS FOR SECTION E,FUNDAMENTAL STEREOCHEMISTRY,” Pure Appl. Chem. 45:13-30, 1976. Desiredenantiomers can be obtained by chiral synthesis from commerciallyavailable chiral starting materials by methods well known in the art, ormay be obtained from mixtures of the enantiomers by separating thedesired enantiomer by using known techniques.

Compounds of this invention may also exhibit geometrical isomerism.Geometrical isomers include the cis and trans forms of compounds of theinvention having alkenyl or alkenylenyl moieties. The present inventioncomprises the individual geometrical isomers and stereoisomers andmixtures thereof.

C. Compound Preparation

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.Unless otherwise indicated, the starting materials are commerciallyavailable and well known in the art. It will be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures) are given, otherprocess conditions can also be used unless otherwise stated. Optimumreaction conditions may vary with the particular reactants or solventused, but such conditions can be determined by one skilled in the art byroutine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and P. G. M. Wuts, Protecting Groups inOrganic Synthesis, Second Edition, Wiley, New York, 1991, and referencescited therein.

Furthermore, the compounds of this invention may contain one or morechiral centers. Accordingly, if desired, such compounds can be preparedor isolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included within the scope ofthis invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents, and the like.

Compounds of the invention can be prepared by methods known in the artand further described herein. For example, methods for making compoundsof formula (I) are described in published application PCT/US2007/084154(WO 2008/063912). Examples of additional synthesis methods applicable tothe preparation of compounds of formula (I) are provided herein.

An example of the preparation of certain KSP inhibitors of Formula I isshown below in Scheme 1.

Butylglycine 1.1 is treated with ethyl chloroformate to form a mixedanhydride 1.2. Benzonitrile 1.3 is treated with ammonium sulfide to formthioamide 1.4, which is them treated with hydrazine to form thehydrazide of formula 1.5. Hydrazide 1.5 reacts with 1.2 andtriethylamine to give carbamate 1.6. Carbamate 1.6 is then refluxed withammonium acetate (NH₄OAc) in xylene to give triazole 1.7. Reaction of1.7 with a fluorinated benzyl bromide ((2-fluorophenyl)methyl bromide or(3-fluorphenyl)methyl bromide) and Cs₂CO₃ in dimethylformamide affords1.8 with its regioisomer (separated from column chromatography).Treatment of 1.8 with trifluoroacetic acid provides the TFA salt of 1.8which can then be converted to its free base when titrated with aNaOH/methanol solution. The formation of 1.8 from 1.1 and 1.3 proceedswith high yield and high purity (>97% as determined by HPLC) and highoptical purity (>99% e.e.) in similar preparations using benzylbromiderather than a fluorinated benzyl bromide.

Compound 1.9 can be reacted with aldehyde 2.7 under reductive aminationconditions to give secondary amine 3.1, which can then be acylated toprovide compounds of formula (I). Scheme 2 illustrates the preparationof aldehyde 2.7 that can be used in the reductive amination step toprepare compounds of formula I, particularly compounds of formula Ia.Cyclic amine 2.1 is protected with Cbz group to give compound 2.2.Epoxide 2.3 is obtained from MCPBA epoxidation of compound 2.2. Epoxidegives racemic mixture of alcohol 2.4 by reacting vinylmagnesium bromideand copper bromide. Alcohol 2.5 as a single enantiomer is obtained bychiral column chromatography. Alcohol 2.5 is subjected to a fluorinationcondition to give vinyl fluoropyrrolidine 2.6. Vinyl fluoropyrrolidine2.6 undergoes subsequent dihydroxylation/oxidative cleavage to givealdehyde 2.7.

After the attachment of the fluoropyrrolidine moiety, known acylatingagents and conditions are used to acylate the acyclic amine to providecompounds of formula (I), after which a protecting group on the nitrogenof the pyrrolidine ring and/or a protecting group of the acylatingmoiety is deprotected. Scheme 3 illustrates the reductive amination toprovide secondary amine 3.1. Acylation of the amine 3.1 followed bydeprotection of the Cbz group and removal of a protecting group on thefree hydroxyl group provides compound 3.4. Suitable protective groupsfor the pyrrolidine ring nitrogen, for example, benzyl carbamates thatcan be removed by hydrogenolysis and t-butyl carbamates that can beselectively removed with reagents such as trimethylsilyl iodide or acid.

Scheme 4 represents general description for urea formation fromsecondary amine 3.1. Secondary amine 3.1 reacts phosgene or triphosgeneto give chlorocompound 4.1, which directly reacts with amine to giveurea compounds of formular (I), after which a protecting group on thenitrogen of the pyrrolidine ring is deprotected. Scheme 4 illustratedthe chlorocarbonylation of secondary amine 3.1 to give compound 4.1,which immediately reacts with morpholine followed by removal of Cbzgroup to give compound 4.2.

Note that the absolute stereochemistry of the chiral centers in thismolecule is identified based on the chirality of known startingmaterials or intermediates. HPLC and nmr data support the conclusionthat the above process provides the compound as a single isomer.

Suitable acylating agents and acids for the acylation step include acylhalides, anhydrides, and acids having the appropriate structures (seeformula I). Suitable amide coupling conditions include use of a varietyof amide coupling reagents to form the amide bond, such as thecarbodiimides N—N′-dicyclohexylcarbodiimide (DCC),N—N′-diisopropylcarbodiimide (DIPCDI), and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI). The carbodiimidesmay be used in conjunction with additives such as dimethylaminopyridine(DMAP) or benzotriazoles such as 7-aza-1-hydroxybenzotriazole (HOAt),1-hydroxybenzotriazole (HOBt), and 6-chloro-1-hydroxybenzotriazole(Cl-HOBt); conditions for such amide bond formations are well known inthe art.

Additional amide coupling reagents also include amininum and phosphoniumbased reagents. Aminium salts includeN-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HBTU),N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HCTU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TBTU), andN-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TCTU). Phosphonium salts includebenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP) andbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP).

The amide formation step may be conducted in a polar solvent such asdimethylformamide (DMF) and may also include an organic base such asdiisopropylethylamine (DIEA) or dimethylaminopyridine (DMAP).

The following compounds in Table 1 were prepared by using one of themethods outlined above. Table I also provides IC50 values for thevarious examples.

D. Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of the subject inventionare usually administered in the form of pharmaceutical compositions.These compositions can be administered by a variety of routes includingoral, parenteral, transdermal, topical, rectal, and intranasal. Thesecompounds are effective, for example, as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of the subjectinvention above associated with pharmaceutically acceptable carriers. Inmaking the compositions of this invention, the active ingredient isusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be in the form of a capsule, sachet,paper or other container. The excipient employed is typically anexcipient suitable for administration to human subjects or othermammals. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The quantity of active component, that is the compound according to thesubject invention, in the pharmaceutical composition and unit dosageform thereof may be varied or adjusted widely depending upon theparticular application, the potency of the particular compound and thedesired concentration.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 1 to about 500 mg, usually about 5 to about100 mg, occasionally about 10 to about 30 mg, of the active ingredient.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Preferably, the compound of thesubject invention above is employed at no more than about 20 weightpercent of the pharmaceutical composition, more preferably no more thanabout 15 weight percent, with the balance being pharmaceutically inertcarrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically or therapeuticallyeffective amount. It will be understood, however, that the amount of thecompound actually administered will be determined by a physician, in thelight of the relevant circumstances, including the condition to betreated, the severity of the condition being treated, the chosen routeof administration, the actual compound administered, the age, weight,and response of the individual patient, the severity of the patient'ssymptoms, and the like.

In therapeutic use for treating, or combating, cancer in mammals, thecompounds or pharmaceutical compositions thereof will be administered byany appropriate route, such as orally, topically, transdermally, and/orparenterally at a dosage to obtain and maintain a concentration, thatis, an amount, or blood-level of active component in the mammalundergoing treatment that will be therapeutically effective. Generally,such therapeutically effective amount of dosage of active component(i.e., an effective dosage) will be in the range of about 0.1 to about100, more preferably about 1.0 to about 50 mg/kg of body weight/day.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mgMicrocrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10%solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch and magnesium stearate are blended, passedthrough a No. 20 mesh U.S. sieve, and filled into hard gelatin capsulesin 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient   25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum  4.0 mg Sodiumcarboxymethyl cellulose (11%)/ 50.0 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to  5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 8

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

Formulation Example 9

A subcutaneous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Formulation Example 11

An illustrative example of an intravenous formulation may be prepared asfollows:

Ingredient Quantity Active Ingredient  250 mg Isotonic saline 1000 mL

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985).

E. Dosage and Administration

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half-life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half-life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each ofwhich is incorporated herein by reference.

Compounds of the instant invention are useful for inhibiting or treatinga disorder mediated, at least in part, by the activity of KSP. In oneaspect, the disorder that is mediated, at least in part by KSP, is acellular proliferative disorder. The term “cellular proliferativedisorder” or “cell proliferative disorder” refers to diseases including,for example, cancer, tumor, hyperplasia, restenosis, cardiachypertrophy, immune disorder and inflammation. The present inventionprovides methods of treating a human or mammalian subject in need ofsuch treatment, comprising administering to the subject atherapeutically effective amount of a compound of formula I or II,either alone or in combination with other anticancer agents.

The compounds of the invention are useful in vitro or in vivo ininhibiting the growth of cancer cells. The term “cancer” refers tocancer diseases including, for example, lung and bronchus; prostate;breast; pancreas; colon and rectum; thyroid; stomach; liver andintrahepatic bile duct; kidney and renal pelvis; urinary bladder;uterine corpus; uterine cervix; ovary; multiple myeloma; esophagus;acute myelogenous leukemia; chronic myelognous leukemia; lymphocyticleukemia; myeloid leukemia; brain; oral cavity and pharynx; larynx;small intestine; non-hodgkin lymphoma; melanoma; and villous colonadenoma.

Cancer also includes tumors or neoplasms selected from the groupconsisting of carcinomas, adenocarcinomas, sarcomas, and hematologicalmalignancies.

Additionally, the type of cancer can be selected from the groupconsisting of growth of solid tumors/malignancies, myxoid and round cellcarcinoma, locally advanced tumors, human soft tissue carcinoma, cancermetastases, squamous cell carcinoma, esophageal squamous cell carcinoma,oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producingtumors, nonsmall cell cancers, breast cancer, gastrointestinal cancers,urological cancers, malignancies of the female genital tract,malignancies of the male genital tract, kidney cancer, brain cancer,bone cancers, skin cancers, thyroid cancer, retinoblastoma,neuroblastoma, peritoneal effusion, malignant pleural effusion,mesothelioma, Wilms's tumors, gall bladder cancer, trophoblasticneoplasms, hemangiopericytoma, and Kaposi's sarcoma.

A compound or composition of this invention may be administered to amammal by a suitable route, such as orally, intravenously, parenterally,transdermally, topically, rectally, or intranasally.

Mammals include, for example, humans and other primates, pet orcompanion animals, such as dogs and cats, laboratory animals, such asrats, mice and rabbits, and farm animals, such as horses, pigs, sheep,and cattle.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant” and can lead todeath of the organism. Malignant neoplasms or “cancers” aredistinguished from benign growths in that, in addition to exhibitingaggressive cellular proliferation, they can invade surrounding tissuesand metastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater“dedifferentiation”) and organization relative to one another and tosurrounding tissues. This property is called “anaplasia.”

Compounds having the desired biological activity may be modified asnecessary to provide desired properties such as improved pharmacologicalproperties (e.g., in vivo stability, bio-availability), or the abilityto be detected in diagnostic applications. Stability can be assayed in avariety of ways such as by measuring the half-life of the compoundsduring incubation with peptidases or human plasma or serum.

For diagnostic purposes, a wide variety of labels may be linked to thecompounds, which may provide, directly or indirectly, a detectablesignal. Thus, the compounds and/or compositions of the subject inventionmay be modified in a variety of ways for a variety of end purposes whilestill retaining biological activity. In addition, various reactive sitesmay be introduced for linking to particles, solid substrates,macromolecules, and the like.

Labeled compounds can be used in a variety of in vivo or in vitroapplications. A wide variety of labels may be employed, such asradionuclides (e.g., gamma-emitting radioisotopes such as technetium-99or indium-111), fluorescers (e.g., fluorescein), enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chemiluminescentcompounds, bioluminescent compounds, and the like. Those of ordinaryskill in the art will know of other suitable labels for binding to thecomplexes, or will be able to ascertain such using routineexperimentation. The binding of these labels is achieved using standardtechniques common to those of ordinary skill in the art.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

The amount administered to the patient will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from a disease in anamount sufficient to cure or at least partially arrest the progressionor symptoms of the disease and its complications. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on the disease condition beingtreated as well as by the judgment of the attending clinician dependingupon factors such as the severity of the disease, disorder or condition,the age, weight and general condition of the patient, and the like.

The compounds administered to a patient are typically in the form ofpharmaceutical compositions described above. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of the compoundpreparations typically will be between about 3 and 11, more preferablyfrom about 5 to 9 and most preferably from about 7 to 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds and/or compositions of thepresent invention will vary according to, for example, the particularuse for which the treatment is made, the manner of administration of thecompound, the health and condition of the patient, and the judgment ofthe prescribing physician. For example, for oral administration, thedose will typically be in the range of about 5 μg to about 50 mg perkilogram body weight per day, preferably about 1 mg to about 10 mg perkilogram body weight per day. In the alternative, for intravenousadministration, the dose will typically be in the range of about 5 μg toabout 50 mg per kilogram body weight, preferably about 500 μg to about5000 μg per kilogram body weight. Alternative routes of administrationcontemplated include, but are not limited to, intranasal, transdermal,inhaled, subcutaneous and intramuscular. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

In general, the compounds and/or compositions of the subject inventionwill be administered in a therapeutically effective amount by any of theaccepted modes of administration for agents that serve similarutilities. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices arepreferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compoundand/or composition used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range which includes the IC₅₀ (theconcentration of the test compound which achieves a half-maximalinhibition of activity) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

EXAMPLES

Referring to the examples that follow, compounds of the presentinvention were synthesized using the methods described herein, or othermethods, which are well known in the art. It is understood thatcompounds not prepared or analyzed may be prepared or analyzed using themethods described herein, or other methods, which are well known in theart.

The compounds and/or intermediates were characterized by highperformance liquid chromatography (HPLC) using a Waters Milleniumchromatography system with a 2690 Separation Module (Milford, Mass.).The analytical columns were Alltima C-18 reversed phase, 4.6×250 mm fromAlltech (Deerfield, Ill.). A gradient elution was used, typicallystarting with 5% acetonitrile/95% water and progressing to 100%acetonitrile over a period of 40 minutes. All solvents contained 0.1%trifluoroacetic acid (TFA). Compounds were detected by ultraviolet light(UV) absorption at either 220 or 254 nm. HPLC solvents were from Burdickand Jackson (Muskegan, Mich.), or Fisher Scientific (Pittsburgh, Pa.).In some instances, purity was assessed by thin layer chromatography(TLC) using glass or plastic backed silica gel plates, such as, forexample, Baker-Flex Silica Gel 1B2-F flexible sheets. TLC results werereadily detected visually under ultraviolet light, or by employing wellknown iodine vapor and other various staining techniques.

Mass spectrometric analysis was performed on one of two LC/MSinstruments: a Waters System (Alliance HT HPLC and a Micromass ZQ massspectrometer; Column. Eclipse XDB-C18, 2.1×50 mm; solvent system: 5-95%(or 35-95%, or 65-95% or 95-95%) acetonitrile in water with 0.05% TFA;flow rate 0.8 mL/min; molecular weight range 500-1500; cone Voltage 20V; column temperature 40° C.) or a Hewlett Packard System (Series 1100HPLC; Column: Eclipse XDB-C18, 2.1×50 mm; solvent system: 1-95%acetonitrile in water with 0.05% TFA; flow rate 0.4 mL/min; molecularweight range 150-850; cone Voltage 50 V; column temperature 30° C.). Allmasses were reported as those of the protonated parent ions.

GC/MS analysis is performed on a Hewlett Packard instrument (HP6890Series gas chromatograph with a Mass Selective Detector 5973; injectorvolume: 1 mL; initial column temperature: 50° C.; final columntemperature: 250° C.; ramp time: 20 minutes; gas flow rate: 1 mL/min;column: 5% phenyl methyl siloxane, Model No. HP 190915-443, dimensions:30.0 m×25 m×0.25 m).

Nuclear magnetic resonance (NMR) analysis was performed on some of thecompounds with a Varian 300 MHz NMR (Palo Alto, Calif.). The spectralreference was either TMS or the known chemical shift of the solvent.Some compound samples were run at elevated temperatures (e.g., 75° C.)to promote increased sample solubility.

The purity of some of the invention compounds is assessed by elementalanalysis (Desert Analytics, Tucson, Ariz.).

Melting points are determined on a Laboratory Devices MeI-Temp apparatus(Holliston, Mass.).

Preparative separations were carried out using a Flash 40 chromatographysystem and KP-Sil, 60A (Biotage, Charlottesville, Va.), or by flashcolumn chromatography using silica gel (230-400 mesh) packing material,or by HPLC using a C-18 reversed phase column. Typical solvents employedfor the Flash 40 Biotage system and flash column chromatography weredichloromethane, methanol, EtOAc, hexane, acetone, aqueous hydroxyamineand triethyl amine Typical solvents employed for the reverse phase HPLCwere varying concentrations of acetonitrile and water with 0.1%trifluoroacetic acid.

Unless otherwise stated, all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

-   AcOH=acetic acid-   aq.=aqueous-   ATP=adenosine triphosphate-   Boc=tert-butyloxycarbonyl-   BSA=bovine serum albumin-   CAM=ceric ammonium molybdate-   DCM=dichloromethane-   DIAD=diisopropyl azodicarboxylate-   DIBAL=diisobutylaluminum hydride-   DIEA=diisopropylethylamine-   DIPEA=diisopropylethylamine-   DMAP=dimethylaminopyridine-   DMF=dimethylformamide-   DMSO=dimethylsulfoxide-   DTT=dithiothreitol-   eq.=equivalents-   Et2O=diethyl ether-   Et₃N=triethyl amine-   EtOAc=ethyl acetate-   EtOH=ethanol-   g=gram-   h=hour-   HPLC=high performance liquid chromatography-   L=liter-   LC/MS=liquid chromatography/mass spectroscopy-   M=molar-   m=meter-   m/z=mass/charge ratio-   MeNH2=methyl amine-   mg=milligram-   min=minute-   mL=milliliter-   mm=millimeter-   mM=millimolar-   mmol=millimole-   mol=mole-   N=normal-   nm=nanometer-   nM=nanomolar-   NMR=nuclear magnetic resonance-   PPh3=triphenyl phosphine-   PhCF3=trifluoromethylbenzene-   psi=pounds per square inch-   RT=room temperature-   sat.=saturated-   TEA=triethylamine-   THF=tetrahydrofuran-   TFA=trifluoroacetic acid-   TLC=thin layer chromatography-   TMS=trimethylsilyl-   TMSCl=trimethylsilyl chloride-   μg=microgram-   μL=microliter-   μM=Micromolar-   Uplc=Ultra performance liquid chromatography

Example 1(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide

Procedure Cbz protection of 2,5-dihydro-1H-pyrrole

To a solution of 2,5-dihydro-1H-pyrrole (30 g, 434 mmol, 96% from AlfaAesar) in dioxane (1000 mL, 0.43 M solution) was added CbzOSu (130 g,521 mmol). After being stirred at room temperature for 18 h, thereaction mixture was concentrated to around 300 mL, diluted with 1000 mLof EtOAc. The organic layer was washed with water and brine, dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo. The desiredbenzyl 2,5-dihydro-1H-pyrrole-1-carboxylate was obtained in 91% yield(80.0 g) as a colorless oil by flash column chromatography. Rf=0.6 (30%EtOAc in hexanes). ¹H NMR (CDCl₃, 400 MHz): δ 7.32 (5H, m), 5.80 (2H,m), 5.77 (2H, s), 4.22 (4H, m). LC/MS (uplc): MH⁺ 204.2, 160.1 (−44),0.86 min.

Epoxidation of benzyl 2,5-dihydro-1H-pyrrole-1-carboxylate

To a solution of benzyl 2,5-dihydro-1H-pyrrole-1-carboxylate (33 g, 163mmol; 90% from Aldrich) in dichloromethane (540 mL, 0.3 M solution) wasadded MCPBA (44 g, 340 mmol, 77% from Aldrich). After the reactionmixture was stirred at room temperature for 18 h, 500 mL of saturatedNa₂CO₃ aqueous solution was added and the resulting mixture was stirredat room temperature for 1 h. The organic layer was separated, washedwith water and brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo. The desired product as a yellow oil was obtainedin 83% yield (29.5 g) by flash column chromatography. ¹H NMR (CDCl₃, 400MHz): δ 3.38 (2H, m), 3.68 (2H, m), 3.87 (2H, m), 5.11 (2H, s), 7.33(5H, m). LC/MS (uplc): MH⁺ 220.0, 0.69 min.

Ring opening of benzyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate

To a solution of benzyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate(28.5 g, 130 mmol) and CuBr.SMe₂ (26.7 g, 130 mmol) in anhydrous THF(260 mL, 0.5 M solution) at −40° C. was slowly added vinyl magnesiumbromide (520 mL, 1.0 M solution in THF). The reaction mixture was thenwarmed up to −20° C. for 2 h. After quenched with saturated NH₄Claqueous solution (200 mL), the reaction mixture was extracted with EtOAc(500 mL). The organic layer was washed with water and brine, dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. The desiredracemic mixture of trans-(±)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate was obtained in 48% yield(15.5 g) as a yellow oil by flash column chromatography. Rf=0.2 (30%EtOAc in hexanes). ¹H NMR (CDCl₃, 400 MHz): δ 2.71 (1H, m), 3.28 (2H,m), 3.72 (2H, m), 4.11 (1H, m), 5.14 (2H, s), 5.16-5.23 (2H, m), 5.69(1H, m), 7.33 (5H, m). LC/MS (uplc): MH⁺ 248.0, 0.78 min.

Resolution of trans-(±)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate

The racemic mixture of trans-(±)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate (14 g) was resolved by usingchiral HPLC (Chiralpak AD-H Heptane:EtOH:MeOH, 8:1:1). The desiredenantiomerically enriched (3S,4R)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate (6.7 min; 6.3 g, >99.5% ee)and undesired (3R,4S)-benzyl 3-hydroxy-4-vinylpyrrolidine-1-carboxylate(9.3 min; 6.7 g, 99.5% ee) were obtained with 92% recovery.

Fluorination of (3S,4R)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate

To a solution of (3S,4R)-benzyl3-hydroxy-4-vinylpyrrolidine-1-carboxylate (5.0 g, 20.2 mmol) in PhCF₃(81 mL, 0.25 M solution) was added N,N-diisopropylethylamine (53 mL, 303mmol), triethylamine trihydrofluoride (19.8 mL, 121 mmol) andperfluoro-1-butanesulfonyl fluoride (PBSF, 3.6 mL, 20.2 mmol). Theresulting mixture was stirred at room temperature. After 60 and 120minutes, additional perfluoro-1-butanesulfonyl fluoride (3.6 mL, 20.2mmol) was added. After 18 hours, the reaction mixture was transferred toa separatory funnel and was washed twice with 50 mL of 1.0 N HCl(Caution! lots of heat produced), twice with saturated NaHCO₃ aqueoussolution, and once with H₂O and brine. The organic phase was dried overanhydrous Na₂SO₄, filtered and concentrated to provide a crude brownoil. The pure (3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylatewas obtained in 81% yield (4.1 g) as a yellow oil by flash columnchromatography (SiO₂, 10%-30% EtOAc in hexanes). Rf=0.55 (30% EtOAc inhexanes). ¹H NMR (CDCl₃, 400 MHz): δ 7.37-7.25 (5H, m), 5.9 (1H, m),5.24 (2H, m), 5.14 (2H, m), 5.03 (1H, dt, J=52.8, 3.2 Hz), 3.9-3.5 (3H,m), 3.53 (1H, m), 2.83 (1H, m). ¹³C NMR (CDCl₃, 100 MHz): δ 154.7,154.6, 136.6, 131.89, 131.83, 128.48, 128.02, 127.94, 119.00, 118.94,95.23, 94.47, 93.42, 92.67, 66.99, 66.94, 53.16, 52.94, 52.83, 52.60,48.17, 48.02, 47.91, 47.83, 47.2, 47.1. LC/MS (uplc): MH⁺ 250.0, 0.93min.

Oxidative Cleavage of Vinyl Fluoropyrrolidine

To a solution of (3R,4R)-benzyl3-fluoro-4-vinylpyrrolidine-1-carboxylate (1.78 g, 7.15 mmol) in CH₃OHand H₂O (2:1, 30 mL, 0.2 M solution) was added a solution of OsO₄ in H₂O(3 mL of a 4% w/v solution, 0.5 mmol). NaIO₄ (4.6 g, 21.5 mmol) was thenadded in a single portion and the resulting mixture was stirred at roomtemperature. After 2 hours, the mixture was filtered to removeprecipitated white solids and the filter cake was washed with EtOAc. Thefiltrate was concentrated in vacuo to remove the majority of the organicsolvents. The residue was extracted with three portions of EtOAc and thecombined organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude (3R,4S)-benzyl3-fluoro-4-formylpyrrolidine-1-carboxylate was used for the next stepwithout further purification. LC/MS (uplc): MH⁺ 208.2 (−44), 252.0, 0.69min.

Synthesis of 2,5-difluorobenzothioamide

A stirred solution of 2,5-difluorobenzonitrile (25 g, 180 mmol) inpyridine (90 mL) was treated with 20 wt % ammonium sulfide in water(67.4 mL, 198 mmol) and triethylamine (27.4 mL, 198 mmol). The reactionmixture was stirred at 50° C. for 5 hr until the reaction was complete.After cooling to room temperature, the mixture was diluted with coldwater and extracted with EtOAc. The organic layer was separated, thenwashed with H₂0 (×3), brine (×3), then dried over anhydrous Na₂SO₄,filtered, and evaporated under reduced pressure to give the crudeproduct. Purification on silica gel column (20% EtOAc in hexanes) toafford 2,5-difluorobenzothioamide as yellow solid (31.0 g, 99%). ¹H NMR(CDCl₃, 300 MHz): δ 7.12 (m, 2H), 7.90 (br, 2H), 8.08 (m, 1H). LC/MS(uplc): MH⁺ 174.0, 0.64 min.

Synthesis of 2,5-difluorobenzimidohydrazide

To a stirred solution of 2,5-difluorobenzothioamide (22.5 g, 129.7 mmol)in EtOH (150 mL) was added hydrazine (6.1 mL, 194.5 mmol). Afterstirring at room temperature for 30 min, reaction was complete by LC/MSand white solid precipitated. The precipitate was filtered and washedwith Hexanes to afford 2,5-difluorobenzimidohydrazide (5.52 g, 94%).

Acylation of 2,5-difluorobenzimidohydrazide

The N-Boc-D-tert-butylglycine (7.5 g, 32.4 mmol) was converted to amixed anhydride by adding ethyl chloroformate (3.41 mL, 35.6 mmol), Et₃N(6.8 mL, 48.6 mmol) in anhydrous THF (65 mL, 0.5 M) at −5° C. to 0° C.The mixture was stirred at −5° C. for 30 min. The resulting solid wasfiltered off and additional anhydrous THF was added to wash theprecipitate. The resulting reaction solution was then added to a THFsolution of 2,5-difluorobenzimido-hydrazide (5.53 g, 32.4 mmol) at −5°C. Then the reaction was gradually warmed to room temperature andstirred for overnight. Once the reaction was complete, the mixture waspartitioned between EtOAc and H₂O. The organic layer was separated andwashed with H₂O, brine, then dried over anhydrous Na₂SO₄, filtered, andevaporated under reduced pressure to give the crude product, which waspurified on silica gel column (50% EtOAc in Hexanes) to afford(R)-tert-butyl1-(2-((2,5-difluorophenyl)(imino)methyl)hydrazinyl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate(67%). Rf=0.4 (50% EtOAc in Hexanes). LC/MS (uplc): MH⁺ 385.3, 0.65 min.

Synthesis of (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate

(R)-tert-Butyl1-(2-((2,5-difluorophenyl)(imino)methyl)hydrazinyl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate(8.35 g, 21.7 mmol) was dissolved in xylenes (200 mL). A Dean-Stark trapwas equipped and the reaction mixture was heated to 150° C. Once thereaction was complete, the mixture was allowed to cool to roomtemperature, then partitioned between EtOAc and saturated aqueous NaHCO₃solution. The organic layer was separated, then washed with saturatedaqueous NaHCO₃ solution, H₂O, and brine, then dried over Na₂SO₄,filtered, and evaporated under reduced pressure to give (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(7.81 g, 98%), which was used for the next step without furtherpurification. LC/MS (uplc): MH⁺ 367.2, 0.98 min.

Alkyation of (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamatewith benzyl bromide

To a stirred suspension of (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(5.89 g, 16.1 mmol) and Cs₂CO₃ (10.5 g, 32.2 mmol) in DMF (46 mL, 0.35M) was added benzyl bromide (2.11 mL, 17.7 mmol). Once the The organiclayer was separated and washed with H₂O, brine, then dried over Na₂SO₄,filtered, and evaporated in vacuo to give (R)-tert-butyl1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl-carbamate.The desired regioisomer was obtained on silica gel column (0% to 100%EtOAc in Hexanes, 3.25 g, 44.3%). The structure was verified by ¹H NMRnOe experiments.

For the desired isomer, ((R)-tert-butyl1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate):crystals, LC/MS (uplc): MH⁺ 457.2, 1.36 min. ¹H NMR (CDCl₃, 300 MHz): δ7.78 (m, 1H), 7.29-7.39 (m, 5H), 7.00-7.18 (m, 2H), 5.53 (s, 2H), 5.20(d, 2H), 4.83 (m, 2H), 1.41 (s, 9H), 0.91 (s, 9H). For the undesiredisomer, ((R)-tert-butyl1-(1-benzyl-5-(2,5-difluorophenyl)-1H-1,2,4-triazol-3-yl)-2,2-dimethylpropylcarbamate):colorless oil, LC/MS (uplc): MH⁺ 457.2, 1.25 min. ¹H NMR (CDCl₃, 300MHz): δ 7.25 (m, 5H), 7.15 (m, 2H), 7.05 (m, 1H), 5.45 (d, 2H), 5.28 (s,2H), 4.85 (d, 2H), 1.43 (s, 9H), 0.97 (s, 9H).

Deprotection of (R)-tert-butyl1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate

(R)-tert-Butyl1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(3.25 g, 7.13 mmol) was treated with TFA (10 mL) in CH₂Cl₂ (30 ml). Oncethe reaction was complete, the reaction was concentrated in vacuo andthen partitioned between EtOAc and saturated aqueous NaHCO₃ solution.The organics were separated, then washed with H₂O, brine, then driedover Na₂SO₄, filtered, and evaporated under reduced pressure to give(R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropan-1-aminewhich was directly used for the next step without further purification(2.32 g, 91%). LC/MS (uplc): MH⁺ 357.1, 0.82 min.

Reductive Alkylation

To a solution of(R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropan-1-amine(2.55 g, 7.15 mmol) in CH₂Cl₂ (59 mL) was added the crude (3R,4S)-benzyl3-fluoro-4-formylpyrrolidine-1-carboxylate (obtained from 1.4 equiv. of(3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylate) in CH₂Cl₂ (10mL) and NaBH(OAc)₃ (2.3 g, 10.7 mmol). The reaction mixture was thenstirred for 16 h at room temperature. After quenched with saturatedNaHCO₃ aqueous solution, the reaction mixture was extracted with EtOAc.The organic layer was washed with water and brine, dried over anhydrousNa₂SO₄, filtered and concentrated. The crude reductive amination productwas contaminated by the HF eliminated alkene product, which was notseparable on either silica column chromatography or preparative reversephase HPLC. Therefore, the crude mixture (3.0 g) was dissolved inacetone and water (5:1, 120 mL). 4-Methylmorpholine N-oxide (715 mg, 6.1mmol) and OsO₄ (2.15 mL of a 4% w/v solution) were added to thisreaction mixture, which was then stirred for over the weekend at roomtemperature. After removal of acetone in vacuo, the remaining aqueouslayer was extracted with EtOAc. The organic layer was washed with waterand brine, dried over anhydrous Na₂SO₄, filtered and concentrated. Thedesired (3R,4R)-benzyl3-(((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylatewas obtained on silica column chromatography (35% to 80% EtOAc inHexanes, 1.5 g, 35%). LC/MS (uplc) MH+ 592.3, 0.97 min.

Reductive Alkylation

To a solution of(R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropan-1-amine(2.55 g, 7.15 mmol) in CH₂Cl₂ (59 mL) was added the crude (3R,4S)-benzyl3-fluoro-4-formylpyrrolidine-1-carboxylate (obtained from 1.4 equiv. of(3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylate) in CH₂Cl₂ (10mL) and NaBH(OAc)₃ (2.3 g, 10.7 mmol). The reaction mixture was thenstirred for 16 h at room temperature. After quenched with saturatedNaHCO₃ aqueous solution, the reaction mixture was extracted with EtOAc.The organic layer was washed with water and brine, dried over anhydrousNa₂SO₄, filtered and concentrated. The crude reductive amination productwas contaminated by the HF eliminated alkene product, which was notseparable on either silica column chromatography or preparative reversephase HPLC. Therefore, the crude mixture (3.0 g) was dissolved inacetone and water (5:1, 120 mL). 4-Methylmorpholine N-oxide (715 mg, 6.1mmol) and OsO₄ (2.15 mL of a 4% w/v solution) were added to thisreaction mixture, which was then stirred for over the weekend at roomtemperature. After removal of acetone in vacuo, the remaining aqueouslayer was extracted with EtOAc. The organic layer was washed with waterand brine, dried over anhydrous Na₂SO₄, filtered and concentrated. Thedesired (3R,4R)-benzyl3-(((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylatewas obtained on silica column chromatography (35% to 80% EtOAc inHexanes, 1.5 g, 35%). LC/MS (uplc) MH+ 592.3, 0.97 min.

Preparation of Acid Chloride

(S)-Tetrahydrofuran-2-carboxylic acid (5.1 g, 44 mmol) was dissolvedwith SOCl₂ (15 mL). The reaction mixture was refluxed for 30 min. Aftervolatile material was removed in vacuo, the crude(S)-tetrahydrofuran-2-carbonyl chloride (6.0 g, >99%) was used for thenext step.

Amide Bond Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(500 mg, 0.845 mmol) in dichloromethane (8.5 mL, 0.1 M solution) at roomtemperature was added triethylamine (236 μL, 1.69 mmol). The crude(S)-tetrahydrofuran-2-carbonyl chloride (227 mg, 1.69 mmol) was thenadded dropwise over 2 min. The resulting solution was stirred at roomtemperature for overnight. The reaction mixture was quenched with H₂Oand extracted with EtOAc. The organic layer was dried over anhydroussodium sulfate and filtered. After the volatile organic materials wereremoved in vacuo, the desired (3R,4R)-benzyl3-(((S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)tetrahydrofuran-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylatewas purified on flash column chromatography (yield: N/A) 0%-100%, EtOAcin hexanes). LC/MS (uplc): MH⁺ 690.5, 1.35 min.

Deprotection

To a solution of (3R,4R)-benzyl3-(((S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)tetrahydrofuran-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(583 mg, 0.845 mmol) in degassed EtOAc (8 mL, 0.1 M solution) was addedPd/C (899 mg, 10 wt %) under anhydrous N₂ atmosphere. After flushed withhydrogen gas, the reaction mixture equipped with a hydrogen gas balloonwas stirred at room temperature for overnight. The reaction mixture wasfiltered through Celite® pad that was washed with EtOAc. The volatileorganic filtrate was removed in vacuo to give the crude product, whichwas purified by preparative reverse phase HPLC. The combined fractionsof the product were neutralized with saturated NaHCO₃ solution, whichwas then extracted with EtOAc. The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated in vacuo. The driedproduct was then dissolved in acetonitrile and water (1:1 ratio) andlyophilized for 48 h. The white powdery(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamidewas obtained in 27.9% yield (131 mg) as a free amine ¹H NMR (CD₃Cl, 400MHz): δ 7.82 (1H, m), 7.53 (2H, m), 7.35-7.27 (3H, m), 7.14 (1H, m),7.06 (1H, m), 6.14 (1H, s), 5.45 (2H, m), 4.85 (2H, m), 4.72 (1H, m),4.21 (1H, m), 3.97 (1H, m), 3.83 (1H, m), 3.02 (1H, m), 2.80 (1H, m),2.33-1.80 (6H, m), 1.25 (1H, s), 1.01 (1H, m), 0.99 (9H, s). LC/MS(uplc): MH⁺ 556.4, 0.99 min.

Example 2N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide

Procedure Urea Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(110 mg, 0.186 mmol) in dichloromethane (1.9 mL, 0.1 M solution) wasadded triethylamine (78 μL, 0.558 mmol) followed by addition of 20%phosgene in toluene (66 mg, 0.223 mmol) at room temperature. After theresulting solution was stirred at room temperature for 15 min (LC/MS(uplc): MH⁺ 654.3, 1.37 min for (3S,4R)-benzyl3-((((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)(chlorocarbonyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate),2,6-dimethylmorpholine (64 mg, 0.558 mmol) was added and heat at 40° C.for overnight (in a seal tube). The reaction mixture was quenched withH₂O and extracted with EtOAc. The organic layer was washed with NaHCO₃solution and brine, dried over anhydrous sodium sulfate, filtered andconcentrated. After the volatile organic materials were removed invacuo, the crude product was purified on preparative reverse phase HPLC.The combined fractions of the product were neutralized with saturatedNaHCO₃ solution, which was then extracted with EtOAc. The organic layerwas dried over anhydrous sodium sulfate, filtered and concentrated invacuo to yield the desired (3R,4R)-benzyl3-(((2S,6R)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-2,6-dimethylmorpholine-4-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(91 mg, 66.7%, over 2 steps). LC/MS (uplc): MH⁺ 733.5, 1.41 min.

Deprotection

To a solution of (3R,4R)-benzyl3-(((2S,6R)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-2,6-dimethylmorpholine-4-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(91 mg, 0.124 mmol) in degassed ethanol (12 mL, 0.1 M solution) wasadded Pd/C (2.64 mg, 10 wt %) under anhydrous N₂ atmosphere. Afterflushed with hydrogen gas, the reaction mixture equipped with a hydrogengas balloon was stirred at room temperature for 45 min. The reactionmixture was filtered through Celite® pad that was washed with EtOAc. Thevolatile organic filtrate was removed in vacuo to give the crudeproduct, which was purified by preparative reverse phase HPLC. Thecombined fractions of the product were neutralized with saturated NaHCO₃solution, which was then extracted with EtOAc. The organic layer wasdried over anhydrous sodium sulfate, filtered and concentrated in vacuo.The dried product was then dissolved in acetonitrile and water (1:1ratio) and lyophilized for 48 h. The white powdery(2S,6R)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamidewas obtained in 94% yield (70 mg) as a free amine ¹H NMR (CD₃Cl, 300MHz): δ 7.83 (1H, m), 7.52 (2H, m), 7.34-7.27 (3H, m), 7.08 (1H, m),7.04 (1H, m), 5.94 (2H, m), 5.41 (1H, s), 3.92-3.62 (3H, m), 3.60-3.45(3H, m), 3.17-2.58 (3H, m), 2.57-2.37 (1H, m), 2.35-2.04 (2H, m), 1.85(2H, m), 1.20 (6H, s), 0.9 (1H, m), 0.8 (9H, s). LC/MS (uplc): MH⁺599.5, 1.06 min.

Example 3N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide

Procedure Alkyation of (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamatewith 3-fluorobenzyl bromide (for CHIR782903)

To a stirred suspension of (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(5.89 g, 16.1 mmol) and Cs₂CO₃ (10.5 g, 32.2 mmol) in DMF (46 mL, 0.35M) was added 3-fluorobenzyl bromide (2.17 mL, 17.7 mmol). Once thereaction was complete, the mixture was partitioned between EtOAc andH₂O. The organic layer was separated and washed with H₂O, brine, thendried over Na₂SO₄, filtered, and evaporated in vacuo to give(R)-tert-butyl1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate.The desired less polar regioisomer was obtained by silica gel column (0%to 100% EtOAc in Hexanes, 5.05 g, 66.2%).

For the desired isomer, (R)-tert-butyl1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate:LC/MS (uplc) 475.2, 1.35 min. For the undesired isomer (R)-tert-butyl1-(5-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-3-yl)-2,2-dimethylpropylcarbamate.LC/MS (uplc) MH+ 475.2, 1.24 min.

Deprotection of (R)-tert-butyl1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(for CHIR782903)

(R)-tert-butyl1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylcarbamate(5.05 g, 10.7 mmol) was treated with TFA (10 mL) in CH₂Cl₂ (30 ml). Oncethe reaction was complete, the reaction was concentrated in vacuo andthen partitioned between EtOAc and saturated aqueous NaHCO₃ solution.The organics were separated, then washed with H₂O, brine, then driedover Na₂SO₄, filtered, and evaporated under reduced pressure to give(R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropan-1-aminewhich was directly used for the next step without further purification(2.55 g, 64%). LC/MS (uplc) MH+ 375.1, 0.83 min.

Reductive Alkylation

To a solution of(R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropan-1-amine(2.55 g, 6.8 mmol) in CH₂Cl₂ (58 mL) was added the crude (3R,4S)-benzyl3-fluoro-4-formylpyrrolidine-1-carboxylate (obtained from 1.4 equiv. of(3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylate) in CH₂Cl₂ (10mL) and NaBH(OAc)₃ (2.2 g, 10.2 mmol). The reaction mixture was thenstirred for 16 h at room temperature. After quenched with saturatedNaHCO₃ aqueous solution, the reaction mixture was extracted with EtOAc.The organic layer was washed with water and brine, dried over anhydrousNa₂SO₄, filtered and concentrated. The crude reductive amination productwas contaminated by the HF eliminated alkene product, which was notseparable on either silica column chromatography or preparative reversephase HPLC. Therefore, the crude mixture (2.89 g) was dissolved inacetone and water (5:1, 120 mL). 4-Methylmorpholine N-oxide (667 mg, 5.7mmol) and OsO₄ (1.51 mL of a 4% w/v solution) were added to thisreaction mixture, which was then stirred for over the weekend at roomtemperature. After removal of acetone in vacuo, the remaining aqueouslayer was extracted with EtOAc. The organic layer was washed with waterand brine, dried over anhydrous Na₂SO₄, filtered and concentrated. Thedesired (3R,4R)-benzyl3-(((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylatewas obtained on silica column chromatography (40% to 90% EtOAc inHexanes, 2.16 g, 52%). LC/MS (uplc): MH⁺ 610.2, 0.99 min.

Urea Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(30 mg, 0.049 mmol) in dichloromethane (450 μL, 0.1 M solution) wasadded triethylamine (20.6 μL, 0.148 mmol) followed by addition of 20%phosgene in toluene (6.2 μL, 0.059 mmol) at room temperature. After theresulting solution was stirred at room temperature for 15 min (LC/MS(uplc): MH⁺ 654.3, 1.37 min for (3S,4R)-benzyl3-((((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)(chlorocarbonyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate),2,6-dimethylmorpholine (64 mg, 0.558 mmol) was added and stirred for 15min at room temperature. The reaction mixture was quenched with H₂O andextracted with EtOAc. The organic layer was washed with NaHCO₃ solutionand brine, dried over anhydrous sodium sulfate, filtered andconcentrated. After the volatile organic materials were removed invacuo, the crude product was purified on preparative reverse phase HPLC.The combined fractions of the product were neutralized with saturatedNaHCO₃ solution, which was then extracted with EtOAc. The organic layerwas dried over anhydrous sodium sulfate, filtered and concentrated invacuo to yield the desired (3R,4R)-benzyl3-(((2S,6R)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-2,6-dimethylmorpholine-4-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(30 mg, 81%, over 2 steps). LC/MS (uplc): MH⁺ 751.5, 1.39 min.

Deprotection

To a solution of (3R,4R)-benzyl3-(((2S,6R)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-2,6-dimethylmorpholine-4-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(35 mg, 0.047 mmol) in degassed ethanol (10 mL, 4.7 mM solution) wasadded Pd/C (0.99 mg, 10 wt %) under anhydrous N₂ atmosphere. Afterflushed with hydrogen gas, the reaction mixture equipped with a hydrogengas balloon was stirred at room temperature for overnight. The reactionmixture was filtered through Celite® pad that was washed with EtOAc. Thevolatile organic filtrate was removed in vacuo to give the crudeproduct, which was purified by preparative reverse phase HPLC. Thecombined fractions of the product were neutralized with saturated NaHCO₃solution, which was then extracted with EtOAc. The organic layer wasdried over anhydrous sodium sulfate, filtered and concentrated in vacuo.The dried product was then dissolved in acetonitrile and water (1:1ratio) and lyophilized for 48 h. The white powdery(2S,6R)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamidewas obtained in 63% yield (18 mg) as a free amine ¹H NMR (CD₃Cl, 300MHz): δ 7.84 (1H, m), 7.32-6.95 (6H, m), 5.79 (2H, m), 5.36 (1H, s),4.58 (1H, m), 3.91-3.72 (3H, m), 3.65-3.50 (3H, m), 3.04 (1H, m),2.97-2.65 (2H, m), 2.53 (1H, m), 2.36 (1H, m), 2.18 (1H, m), 1.20 (6H,s), 0.9 (1H, m), 0.84 (9H, s). LC/MS (uplc): MH⁺ 617.5, 1.06 min.

Example 4(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

Procedure Amide Bond Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(1.5 g, 2.53 mmol) in dichloromethane (25 mL, 0.1 M solution) at roomtemperature was added triethylamine (458 μL, 3.29 mmol).(5)-1-Chloro-1-oxopropan-2-yl acetate (416 μL, 3.29 mmol) was then addeddropwise over 2 min. The resulting solution was stirred at roomtemperature for overnight. The reaction mixture was quenched with H₂Oand extracted with EtOAc. The organic layer was dried over anhydroussodium sulfate and filtered. After the volatile organic materials wereremoved in vacuo, the desired (3R,4R)-benzyl3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-propanamido)methyl)-4-fluoropyrrolidine-1-carboxylatewas purified on flash column chromatography (0%400%, EtOAc in hexanes).LC/MS (uplc): MH⁺ 706.5, 1.34 min.

Global Deprotection

To a solution of (3R,4R)-benzyl3-(((S)-2-acetoxy-N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate(1.78 g, 2.53 mmol) in degassed EtOAc (25 mL, 0.1 M solution) was addedPd/C (178 mg, 10 wt %) under anhydrous N₂ atmosphere. After flushed withhydrogen gas, the reaction mixture equipped with a hydrogen gas balloonwas stirred at room temperature for overnight. The reaction mixture wasfiltered through Celite® pad that was washed with EtOAc. The volatileorganic filtrate was removed in vacuo to give the crude amine (LC/MS(uplc): MH⁺ 572.4, 1.11 min) Then, the crude product was dissolved inMeOH (168 mL, 0.015 M solution) followed by addition of anhydrouspotassium carbonate (3.5 g, 25 mmol). The reaction was then stirred for15 min at room temperature. After white precipitate was removed byfiltration, the organic filtrate was concentrated in vacuo. The crudeproduct was purified by preparative reverse phase HPLC. The combinedfractions of the product were neutralized with saturated NaHCO₃solution, which was then extracted with EtOAc (200 mL). The organiclayer was dried over anhydrous sodium sulfate, filtered and concentratedin vacuo. The dried product was then dissolved in acetonitrile and water(1:1 ratio) and lyophilized for 48 h. The white powdery(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamidewas obtained in 65.5% yield (876 mg, over 3 steps) as a free amine ¹HNMR (CD₃Cl, 400 MHz): 7.82 (1H, m), 7.52 (2H, m), 7.34-7.25 (3H, m),7.16 (1H, m), 7.07 (1H, m), 6.09 (1H, s), 5.44 (2H, s), 4.61 (2H, m),4.39 (1H, m), 3.78 (1H, m), 3.60 (1H, m), 2.92 (1H, m), 2.56 (1H, m),2.16 (2H, m), 1.44 (3H, m), 1.22 (1H, m), 0.99 (9H, s), 0.42 (1H, m).LC/MS (uplc): MH⁺ 530.3, 1.05 min.

Example 5(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide

Procedure Amide Bond Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(500 mg, 0.82 mmol) in dichloromethane (4 mL, 0.2 M solution) at roomtemperature was added triethylamine (229 μL, 1.64 mmol). The crude(S)-tetrahydrofuran-2-carbonyl chloride (221 mg, 1.64 mmol) was thenadded dropwise over 2 min. The resulting solution was stirred at roomtemperature for overnight. The reaction mixture was quenched with H₂Oand extracted with EtOAc. The organic layer was dried over anhydroussodium sulfate and filtered. After the volatile organic materials wereremoved in vacuo, the desired (3R,4R)-benzyl3-(((S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)tetrahydrofuran-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylatewas purified on flash column chromatography (yield N/A; 0%-100%, EtOAcin hexanes). LC/MS (uplc): MH⁺ 708.4, 1.35 min.

Deprotection

To a solution of (3R,4R)-benzyl3-(((S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)tetrahydrofuran-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate(580 mg, 0.82 mmol) in degassed EtOAc (80 mL, 0.1 M solution) was addedPd/C (873 mg, 10 wt %) under anhydrous N₂ atmosphere. After flushed withhydrogen gas, the reaction mixture equipped with a hydrogen gas balloonwas stirred at room temperature for overnight. The reaction mixture wasfiltered through Celite® pad that was washed with EtOAc. The volatileorganic filtrate was removed in vacuo to give the crude product, whichwas purified by preparative reverse phase HPLC. The combined fractionsof the product were neutralized with saturated NaHCO₃ solution, whichwas then extracted with EtOAc. The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated in vacuo. The driedproduct was then dissolved in acetonitrile and water (1:1 ratio) andlyophilized for 48 h. The white powdery(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamidewas obtained in 10.4% yield (48.9 mg) as a free amine ¹H NMR (CD₃Cl, 400MHz): 7.82 (1H, m), 7.34-7.25 (3H, m), 7.22 (1H, m), 7.15 (1H, m), 7.07(1H, m), 6.98 (1H, m), 6.10 (1H, s), 5.44 (2H, m), 4.86 (2H, m), 4.19(1H, m), 3.96 (1H, m), 3.86 (2H, m), 3.05 (1H, m), 3.05 (1H, m),2.37-1.70 (2H, m), 1.35-1.02 (5H, m), 0.99 (9H, s). LC/MS (uplc): MH⁺574.4, 0.98 min.

Example 6(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

Amide Bond Formation

To a solution of (3R,4R)-benzyl3-(((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropylamino)methyl)-4-fluoropyrrolidine-1-carboxylate(1.48 g, 2.43 mmol) in dichloromethane (25 mL, 0.1 M solution) at roomtemperature was added triethylamine (440 μL, 3.16 mmol).(S)-1-Chloro-1-oxopropan-2-yl acetate (400 μL, 3.16 mmol) was then addeddropwise over 2 min. The resulting solution was stirred at roomtemperature for overnight. The reaction mixture was quenched with H₂Oand extracted with EtOAc. The organic layer was dried over anhydroussodium sulfate and filtered. After the volatile organic materials wereremoved in vacuo, the desired (3R,4R)-benzyl3-(((5)-2-acetoxy-N-((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylatewas purified by flash column chromatography (0%-100%, EtOAc in hexanes).LC/MS (uplc) MH+ 724.4, 1.34 min.

Global Deprotection

To a solution of (3R,4R)-benzyl3-(((S)-2-acetoxy-N-((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate(2.24 g, 3.09 mmol) in degassed EtOAc (25 mL, 0.1 M solution) was addedPd/C (224 mg, 10 wt %) under anhydrous N₂ atmosphere. After flushed withhydrogen gas, the reaction mixture equipped with a hydrogen gas balloonwas stirred at room temperature for overnight. The reaction mixture wasfiltered through Celite® pad that was washed with EtOAc. The volatileorganic filtrate was removed in vacuo to give the crude amine Then, thecrude product was dissolved in MeOH (200 mL, 0.015 M solution) followedby addition of anhydrous potassium carbonate (4.2 g, 30.9 mmol). Thereaction was then stirred for 15 min at room temperature. After whiteprecipitate was removed by filtration, the organic filtrate wasconcentrated in vacuo. The crude product was purified by preparativereverse phase HPLC. The combined fractions of the product wereneutralized with saturated NaHCO₃ solution, which was then extractedwith EtOAc (200 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated in vacuo. The dried product was thendissolved in acetonitrile and water (1:1 ratio) and lyophilized for 48h. The white powdery(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamidewas obtained in 57% yield (962 mg, over 3 steps) as a free amine ¹H NMR(CD₃OD, 400 MHz): 7.81 (1H, m), 7.40-7.16 (5H, m), 7.06 (1H, m), 6.12(1H, s), 5.44 (2H, m), 4.77 (2H, m), 3.87 (1H, m), 2.82 (2H, m), 2.28(1H, m), 2.17 (1H, m), 1.95 (1H, m), 1.43 (3H, m), 1.22 (1H, m), 0.91(9H, s). LC/MS (uplc): MH⁺ 548.3, 0.94 min.

Example 7 Assay for Determining KSP Activity

This example provides a representative in vitro assay for determiningKSP activity in vitro. Purified microtubules obtained from bovine brainwere purchased from Cytoskeleton Inc. (Denver, Colo., USA). The motordomain of human KSP (Eg 5, KNSL1) was cloned, expressed, and purified togreater than 95% homogeneity. Biomol Green was purchased from BIOMOLInternational L.P. (Plymouth Meeting, Pa., USA). The microtubules wererapidly thawed at 37° C. for 10 minutes before making dilution.Microtubules and KSP motor protein (i.e., the KSP motor domain) werediluted in assay buffer (20 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, 10 mM DTTand 0.125% BSA) to a final concentration of 50 μg/mL microtubules and 3nM KSP.

To each well of the testing plate (96-well half area plate) containing1.25 μL of inhibitor or test compound in DMSO (or DMSO only in the caseof controls) were added 25 μL of ATP solution (ATP diluted to aconcentration of 250 μM in assay buffer) and 25 μL of theabove-described microtubule/KSP solution. The plates were incubated atRT for 2 hours. Following incubation, 65 μL of Biomol Green (a malachitegreen-based dye that detects the release of inorganic phosphate) with0.015% Tween 20 was added to each well. The plates were incubated for anadditional 15-20 minutes then the absorbance at 650 nm was determinedusing a Molecular Devices Spectrophotometer. The amount of absorbance at650 nm corresponded to the amount of KSP activity in the samples. TheIC₅₀ of each inhibitor or test compound was then determined based on thedecrease in absorbance at 650 nm at each concentration, via nonlinearregression using either XLFit for Excel or Prism data analysis softwareby GraphPad Software Inc.

Preferred compounds of the invention have a biological activity asmeasured by an IC₅₀ of less than about 1 mM in assay protocols describedin Example 7, with preferred embodiments having biological activity ofless than about 25 μM, with particularly preferred embodiments havingbiological activity of less than about 1000 nM, and with the mostpreferred embodiments having biological activity of less than about 100nM.

Table 1 lists structures, IC₅₀ values, mass spec data, and retentiontime for the illustrative examples representing the present invention.

TABLE 1 Biological retention CMPD Activity time mass # STRUCTURE (IC50)(min) (MH⁺) method 1

0.96 nM 0.99 556.4 LC/MS (uplc) 2

0.83 nM 1.06 599.5 LC/MS (uplc) 3

0.74 nM 1.06 617.5 LC/MS (uplc) 4

0.60 nM 1.05 530.3 LC/MS (uplc) 5

1.46 nM 0.98 574.4 LC/MS (uplc) 6

0.83 nM 0.94 548.3 LC/MS (uplc)

Additional compounds of the invention are in Table 2 below. Thesecompounds were prepared similarly to those described above, andincorporate an alkoxy group in R¹. The alkoxy-containing portion ofthese compounds can be made as illustrated in the following Examples.

Example 9 Methylation of(R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid

To a solution of sodium hydride suspension in mineral oil (60% wt) (3.86g, 96 mmol) in dry THF (60 mL) were added(R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid (7.5g, 32.2 mmol) in THF (50 mL) at 0° C. slowly. After stirring for 1 h atroom temperature, iodomethane (4.31 ml, 38.6 mmol) was added. Thereaction mixture was stirred at room temperature overnight. Afterquenched with water, the reaction mixture was extracted by ether. Theaqueous layers was acidify to pH=3 by adding 6 N HCl, then extracted byEtOAc. The organic layer was washed with brine, dried over anhydrousNa₂SO₄, filtered though Buchner funnel, and concentrated to yield crude(R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-methylbutanoic acid (7 g,28.3 mmol, 88%), which was used in next step reaction withoutpurification. LC/MS (uplc): MH⁺ 160.1, 0.68 min.

Acylation of 2,5-difluorobenzimidohydrazide

The crude (R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-methylbutanoicacid (4 g, 16.2 mmol) was converted to a mixed anhydride by adding Et₃N(3.38 ml, 24.26 mmol), ethyl chloroformate (1.931 g, 17.79 mmol) inanhydrous THF (32.4 ml, 0.5 M) at −5° C. to 0° C. The mixture wasstirred at −5° C. for 30 min. The resulting solid was filtered off andadditional anhydrous THF was added to wash the precipitate. Theresulting reaction solution was then added to a THF solution of2,5-difluorobenzimido-hydrazide (2.77 g, 16.18 mmol) at −5° C. Then thereaction was gradually warmed to room temperature and stirred forovernight. Once the reaction was complete, the mixture was partitionedbetween EtOAc and H₂O. The organic layer was separated and washed withH₂O, brine, then dried over anhydrous Na₂SO₄, filtered, and evaporatedunder reduced pressure to give the crude product, which was purified onsilica gel column (50% EtOAc in Hexanes) to afford (R)-tert-butyl(1-(2-((2,5-difluorophenyl)(imino)methyl)hydrazinyl)-3-methoxy-3-methyl-1-oxobutan-2-yl)carbamate(1 g, 15%). LC/MS (uplc): MH⁺ 401.2, 0.61 min.

Synthesis of (S)-tert-butyl(1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2-methoxy-2-methylpropyl)carbamate

(R)-tert-Butyl1-(2-((2,5-difluorophenyl)(imino)methyl)hydrazinyl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate(1.0 g, 2.497 mmol) was dissolved in xylenes (8 mL). A Dean-Stark trapwas equipped and the reaction mixture was heated to 160° C. Once thereaction was complete, the mixture was allowed to cool to roomtemperature, then partitioned between EtOAc and saturated aqueous NaHCO₃solution. The organic layer was separated, then washed with saturatedaqueous NaHCO₃ solution, H₂O, and brine, then dried over Na₂SO₄,filtered, and evaporated under reduced pressure to give (S)-tert-butyl(1-(3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2-methoxy-2-methylpropyl)carbamate(0.97 g, 98%), which was used for the next step without furtherpurification. LC/MS (uplc): MH⁺ 383.2, 0.9 min.

TABLE 2 KSP IC50 LCMS/ Cmpd # Mol Structure (uM) Rt (min) MH+ 7

0.00123 0.86 578.2 8

0.00149 0.82 552.3 9

0.04941 0.92 618.3 10

0.0009 0.88 590.2 11

0.0014 0.86 560.4 12

0.00087 0.85 564.4 13

0.00069 0.89 572.4 14

0.00234 0.82 534.4NMR data for representative compounds of this type is provided here:

Cmpd No. ¹H NMR data 7 ¹H NMR (CD₃OD, 400 MHz) δ 7.87-7.73 (m, 1H),7.45-7.31 (m, 1H), 7.32-7.11 (m, 4H), 7.11-6.95 (m, 1H), 6.2 (s, 1H),5.61-5.29 (m, 2H), 4.81-4.69 (m, 1H), 4.51-4.3 (m, 1H), 4.29-3.97 (m,3H), 3.95-3.80 (m, 1H), 3.75-3.61 (m, 1H), 3.16 (s, 3H), 2.38-2.21 (m, 1H), 2.17-1.81 (m, 3H), 1.80-1.66 (m, 1H), 1.26-1.16 (m, 1H), 1.15 (s,3H), 1.05 (s, 3H), 0.94-0.73 (m, 1H) 8 ¹H NMR (CD₃OD, 400 MHz) δ7.85-7.71 (m, 1H), 7.45-7.32 (m, 1H), 7.31-7.14 (m, 4H), 7.11-7.00 (m,1H), 6.2 (s, 1H), 5.63-5.34 (m, 2H), 5.6-5.3 (m, 2H), 4.62-4.40 (m, 2H),4.38-4.23 (m, 1H), 4.21-3.98 (m, 2H), 3.69-3.55 (m, 1H), 3.23-3.09 (m,3H), 2.05-1.94 (m, 1H), 1.76-1.56 (m, 1H), 1.50-1.37 (m, 1H), 1.26-1.15(m, 2H), 1.14 (s, 3H), 1.03 (s, 3H), 0.90-0.74 (m, 1H) 10 ¹H NMR (CD₃OD,400 MHz) δ 7.8 (m, 1H), 7.4 (m, 1H), 7.34-7.14 (m, 4H), 7.09 (m, 1H),6.2 (s, 1H), 5.67-5.39 (m, 2H), 4.78 (m, 1H), 4.36-3.99 (m, 3H),3.99-3.80 (m, 1H), 3.55-3.37 (m, 1H), 3.38-3.32 (m, 1H), 3.12 (s, 3H),2.87-2.69 (m, 1H), 2.55-2.22 (m, 2H), 2.22-2.04 (m, 2H), 2.04-1.79 (m,2H), 1.46-1.31 (m, 1H), 1.28-1.10 (m, 4H), 1.10-0.93 (m, 3H) 14 ¹H NMR(CD₃Cl, 400 MHz): δ 7.80-7.68 (m, 1H), 7.4-7.26 (s, 4H), 7.22-6.97 (m,3H), 6.10-5.97 (s, 1H), 5.56-5.25 (m, 2H), 4.68 (m, 1H), 4.30-3.89 (m,3H), 3.86-3.51 (m, 1H), 3.2-3.1 (m, 1H), 3.07 (s, 3H), 1.58-1.43 (m,2H), 1.35 (d, 3H), 1.25 (m, 1H), 1.05 (s, 3H), 0.94 (s, 3H), 0.5 (m, 1H)

Example 8

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated withKSP Inhibitors

Cells are plated in 96-well plates at densities of about 500 cells perwell of a 96-well plate and are allowed to grow for 24 hours. The cellsare then treated with various concentrations of compounds for 72 hours.Then, 100 μl of CellTiter-Glo® solution are added. The CellTiter-Glo®assay measures the amount of ATP present in the well after lysing thecells; the ATP released is used in an enzymatic reaction including theenzyme Luciferease and its substrate Luciferin. The amount of lightemitted is proportional to the amount of ATP, which in turn isproportional to the number of live cells in the well. (see Promegaproduct catalog #G7573, CellTiter-Glo® Luminescent Cell ViabilityAssay). The cells are then incubated in the dark for 30 minutes. Theamount of luminescence is determined for each well using a Wallac Triluxplate reader, which correlates with the number of cells per well. Thenumber of viable cells in the wells that receive only DMSO (0.5%) serveas an indication of 0% inhibition, while wells without cells serve as100% inhibition of cell growth. The compound concentration that resultsin a 50% growth inhibition (GI₅₀) is determined graphically fromsigmoidal dose-response curves of log-transformed dose values versuscell counts (percent of control) at 72 hours of continuous compoundexposure.

The cell lines used are listed below.

The cell proliferation assay is performed as described above.

Cancer Cell Lines

Colo 205—colon carcinoma

-   -   RPMI 1640+10% FBS+1% L-glutamine+1% P/S+1% NaPyr.+Hepes    -   +4.5 g/L Glucose+1% NaBicarb.

MDA 435—breast cancer—high met

-   -   EMEM+10% FBS+1% P/S+1% L-Glutamine+1% NEAA+1% NaPyr+1% vitamins

HCT-15 and HCT116—colon carcinoma

-   -   RPMI 1640+10% FBS+1% L-glutamine+1% P/S

Drug Resistant Cell Lines

KB3.1—colon epidermal carcinoma; parental cell line

-   -   Iscove's+10% FBS+1% L-glutamine+1% P/S

KBV1—p-glycoprotein associated multi-drug resistant cell line

-   -   RPMI 1640+10% FBS+1% L-glutamine+1% P/S+0.2 ug/mL Vinblastine

KB8.5—p-glycoprotein associated multi-drug resistant cell line

-   -   DMEM+10% FBS+1% L-glutamine+1% P/S+10 ng/mL Colchicine

Preferred compounds of the invention have a biological activity asmeasured by a GI₅₀ of less than about 1 mM in assay protocols describedwith some embodiments having biological activity of less than about 25nM, with other embodiments having biological activity of less than about1000 nM, and with still other embodiment having a GI₅₀ of less thanabout 100 nM.

Example 9 Clonogenic Softagar Assay Protocol

Human cancer cells are plated at a density of 3×10⁵ cells per well in a6-well plate. The next day, a compound of interest at a certainconcentration is added to each well. After 24 and 48 hours ofincubation, the cells are harvested, washed and counted. The followingsteps are performed using the Multimek 96 robot. Then, 500 viable cellsper well are plated in a 96-well plate that is coated with PolyHema toprevent attachment of the cells to the bottom of the well. Agarose (3%stock) is melted, diluted in warmed media and added to the cells to afinal concentration of 0.5%. After the soft agar solidified, the platesare incubated at 37° C. for 6 days. Alamar blue dye added to and platesare incubated for an additional 6 hours. The is measured on a platereader and is considered to correlate with the number of colonies formedin soft agar. A cancerous cell is able to grow on the agar and thus willshow an increase in optical density. A reading of decreased opticaldensity means that the cancer cells are being inhibited. It iscontemplated that compounds of this invention will exhibit a decrease inoptical density.

1. A compound of Formula I

wherein: R¹ is selected from C₁₋₆ straight chain alkyl, C₃₋₆ branchedalkyl, and —C₃₋₆ cyclo alkyl; R² is selected from H, and C₁₋₆ straightchain alkyl; R³ represents —(CH₂)₀₋₃ substituted or unsubstitutedpyrrolidinyl; R⁴ is selected from —C(O)—CH₂OH, —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-unsubstituted morpholinyl, and —C(O)-morpholinylsubstituted with up to three alkyl groups; R⁵ is selected fromsubstituted or unsubstituted benzyl, wherein the substituents areselected from Cl, F, Br, and I; and R⁶ is selected from phenylsubstituted with up to three halogen atoms.
 2. (canceled)
 3. (canceled)4. A compound of claim 1, wherein: R¹ is selected from C₃₋₆ branchedalkyl; R² represents H; R³ represents —(CH₂)₁₋₃-substitutedpyrrolidinyl; R⁴ is selected from —C(O)-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, —C(O)-morpholinyl substituted with up to three alkylgroups; R⁵ represents benzyl, or benzyl substituted with up to twofluoro atoms; and R⁶ is selected from phenyl substituted with up to twohalogen atoms.
 5. A compound of claim 1, wherein: R¹ represents t-butyl;R³ represents —(CH₂)-fluoro-pyrrolidinyl; R⁴ is selected from—C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH, —C(O)-2,6-dimethylmorpholinyl; R⁵ represents benzyl, or benzyl substituted with one fluoroatom; and R⁶ is selected from phenyl substituted with up to two fluoroatoms.
 6. A compound of claim 4, wherein: R³ represents—CH₂₋₃-fluoro-pyrrolidinyl; and R⁴ represents —C(O)-2-tetrahydrofuranyl,—C(O)—CH(CH₃)—OH, or —C(O)-2,6-dimethyl morpholinyl.
 7. A compound ofclaim 6, wherein: R³ represents

R⁴ is selected from —C(O)—CH(CH₃)—OH, and


8. (canceled)
 9. A compound according to claim 1, wherein R⁴ is selectedfrom:


10. A compound of claim 1, selected from:N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2,6-dimethylmorpholine-4-carboxamide;(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)tetrahydrofuran-2-carboxamide;(S)—N—((R)-1-(3-(2,5-difluorophenyl)-1-(3-fluorobenzyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide;and(S)—N—((R)-1-(1-benzyl-3-(2,5-difluorophenyl)-1H-1,2,4-triazol-5-yl)-2,2-dimethylpropyl)-N-(((3R,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide;and the pharmaceutically acceptable salts of any of these compounds. 11.(canceled)
 12. A compound of Formula (II):

wherein, R¹ is selected from C₁₋₆ straight chain alkyl, C₃₋₆ branchedalkyl, and —C₃₋₆ cyclo alkyl; R³ represents —(CH₂)₀₋₃-substituted orunsubstituted pyrrolidinyl or C₃₋₅ alkyl substituted with up to threegroups selected from amino and halo; R⁴ is selected from —C(O)—CH₂OH,—C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH, —C(O)-unsubstitutedmorpholinyl, and —C(O)-morpholinyl substituted with up to three alkylgroups; R⁵ is selected from substituted or unsubstituted benzyl, whereinthe substituents are selected from Cl, F, Br, and I; and R⁶ is selectedfrom phenyl substituted with up to three halogen atoms.
 13. The compoundof claim 12, wherein: R¹ is selected from C₃₋₆ branched alkyl, and C₃₋₆cyclo alkyl; R³ represents —(CH₂)₀₋₃-substituted pyrrolidinyl or—CH₂—CH₂—CH(NH₂)—CH₂F; R⁴ is selected from —C(O)—CH₂OH,—C(O)-tetrahydrofuranyl, —C(O)—CH(CH₃)—OH, —C(O)-unsubstitutedmorpholinyl, and —C(O)-morpholinyl substituted with up to three alkylgroups; R⁵ is selected from substituted or unsubstituted benzyl, whereinthe substituents are selected from Cl, F, Br, and I; and R⁶ is selectedfrom phenyl substituted with up to three halogen atoms. 14-19.(canceled)
 20. The compound of claim 12, wherein R⁴ is selected from:


21. (canceled)
 22. A pharmaceutical composition comprising atherapeutically effective amount of a compound of claim 1, and apharmaceutically acceptable carrier.
 23. The composition of claim 22further comprising at least one additional agent for the treatment ofcancer.
 24. The composition of claim 23, wherein the additional agentfor the treatment of cancer is selected from the group consisting ofirinotecan, topotecan, gemcitabine, imatinib, trastuzumab,5-fluorouracil, leucovorin, carboplatin, cisplatin, docetaxel,paclitaxel, tezacitabine, cyclophosphamide, vinca alkaloids,anthracyclines, rituximab, and trastuzumab.
 25. A method of treating adisorder mediated, at least in part, by KSP in a mammalian patientcomprising administering to a mammalian patient in need of suchtreatment a therapeutically effective amount of a compound of claim 1;wherein the disorder is a cancer is selected from the group consistingof lung and bronchus; prostate; breast; pancreas; colon and rectum;thyroid; stomach; liver and intrahepatic bile duct; kidney and renalpelvis; urinary bladder; uterine corpus; uterine cervix; ovary; multiplemyeloma; esophagus; acute myelogenous leukemia; chronic myelognousleukemia; lymphocytic leukemia; myeloid leukemia; brain; oral cavity andpharynx; larynx; small intestine; non-Hodgkin lymphoma; melanoma; andvillous colon adenoma. 26-28. (canceled)
 30. A method for inhibiting KSPin a mammalian patient, wherein said method comprises administering tothe patient an effective KSP-inhibiting amount of a compound of claim 1.31. A method to make a fluorinated pyrrolidine, which comprises reactinga trans-3,4-disubstituted protected pyrrolidine of Formula V

with a fluorination reagent to provide a compound of Formula VI:

 and oxidizing the compound of Formula VI to an aldehyde of Formula VII:

wherein PG represents a protecting group, and R is selected from H andan optionally substituted alkyl or aryl group.
 32. The method of claim31, which further comprises reductive amination of the compound ofFormula VII with a compound of Formula Ia:

wherein: R¹ is selected from C₁₋₆ alkoxy-C₁₋₄-alkyl, C₁₋₆ straight chainalkyl, C₃₋₆ branched alkyl, and —C₃₋₆ cyclo alkyl; R² is selected fromH, and C₁₋₆ straight chain alkyl; R⁵ is selected from substituted orunsubstituted benzyl, wherein the substituents are selected from Cl, F,Br, and I; and R⁶ is selected from phenyl substituted with up to threehalogen atoms; to provide a compound of Formula Ib:

33-34. (canceled)